bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of wild-type signal sequences and their charged variants with model and natural membranes
Authors: Rao N M , Nagaraj R
Published: August 2015

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.
The ethanolamine requirement of keratinocytes for growth is not due to defective synthesis of ethanolamine phosphoacylglycerols by the decarboxylation pathway
Authors: Arthur G , Lu X
Published: August 2015

We have investigated whether the growth requirement of keratinocytes for ethanolamine is due to defective synthesis of ethanolamine phosphoacylglycerols (EPG) via decarboxylation of serine phosphoacylglycerols. Proliferating keratinocytes readily incorporated [3H]ethanolamine into phosphatidylethanolamine (PE) and [3H]serine into phosphatidylserine (PS) and PE. Non-proliferating keratinocytes in ethanolamine-free medium incorporated [3H]glycerol into phosphatidylcholine (PC), PS and PE in decreasing order of label incorporated. The order of decreasing incorporation of glycerol after addition of ethanolamine to the medium was PC > PE > PS. Incubation of non-proliferating keratinocytes with [3H]serine resulted in incorporation of label into PS and PE. The extent of incorporation of [3H]serine into PS in non-proliferating keratinocytes was not less than that in proliferating cells. Addition of ethanolamine to the medium of non-proliferating keratinocytes did not change the quantity of label incorporated into PS, but resulted in a decrease of label incorporated into PE. When cells were prelabelled overnight with [3H]serine and subsequently incubated in medium containing ethanolamine, the loss of label from PS was inhibited relative to that of control cells incubated in medium without ethanolamine. The activity of PS decarboxylase activity in keratinocyte mitochondria was inhibited by phosphoethanolamine and PE, but not by ethanolamine or CDP-ethanolamine. Both proliferating and non-proliferating keratinocytes incorporated [3H]serine into ether-linked ethanolamine phospholipids. Taken together, the above results suggest that (1) both proliferating and non-proliferating keratinocytes are able to synthesize PE and ether-linked ethanolamine phospholipids from serine, and therefore the ethanolamine-requirement of the cells is not due to a defective decarboxylase pathway; (2) any inability of the decarboxylase pathway to meet cellular EPG requirement is not due to decreased synthesis of serine phospholipids; (3) synthesis of PE via decarboxylation, the major route in nonproliferating keratinocytes, appears to decrease when ethanolamine is made available and the CDP-ethanolamine pathway is functioning; (4) phosphoethanolamine and increased PE produced from the CDP-ethanolamine pathway may inhibit PS decarboxylase activity in the cells and provide a means of coordinating the synthesis of PE by the two pathways to prevent excess production.
Deficiency of Na+/K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle and cultured muscle cells of myotonic dystrophy patients
Authors: Benders A A , Timmermans J A , Oosterhof A , Ter Laak H J , van Kuppevelt T H M S M , Wevers R A , Veerkamp J H
Published: August 2015

Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.
Degenerate 87-base-pair tandem repeats create hydrophilic/hydrophobic alternating domains in human mucin peptides mapped to 11p15
Authors: Dufosse J , Porchet N , Audie J P , Guyonnet Duperat V , Laine A , Van-Seuningen I , Marrakchi S , Degand P , Aubert J P
Published: August 2015

A human tracheobronchial lambda gt 11 cDNA library was screened using antiserum prepared against the deglycosylated protein backbone of human tracheobronchial mucins. Two cDNAs, designated JER 28 and 57, obtained from this immunoscreening, were used to isolate two other cDNA clones, JUL 7 and JUL 10, from a human tracheobronchial lambda gt 10 cDNA library. These four clones (561, 1830, 1631 and 991 bp), which mapped to chromosome 11p15, were all found to contain degenerate 87-base-pair tandem repeats which encode non-repetitive peptides. Numerous deletions or insertions in an otherwise virtually perfect 87-base-pair tandem repeat create many shifts in reading frame which completely destroy the repetitive peptide structure. The peptide is composed of alternate hydrophobic and hydrophilic domains which probably differ in the extent to which they are glycosylated. The mRNAs are expressed both in the respiratory and in the digestive tracts. These human mucin probes may be important in assessing the abnormal mucins associated with inflammatory diseases or carcinoma from human mucosae.
Mobilization of Ca2+ by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone in permeabilized insulin-secreting RINm5F cells: evidence for separate uptake and release compartments in inositol 1,4,5-trisphosphate-sensitive Ca2+ pool
Authors: Islam M S , Berggren P O
Published: August 2015

We characterized and directly compared the Ca(2+)-releasing actions of two inhibitors of endoplasmic-reticulum (ER) Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ), in electropermeabilized insulin-secreting RINm5F cells. Ambient free calcium concentration ([Ca2+]) was monitored by Ca(2+)-selective mini-electrodes. After ATP-dependent Ca2+ uptake, thapsigargin and tBuBHQ released Ca2+ with and EC50 of approximately 37 nM and approximately 2 microM respectively. Both agents mobilized Ca2+ predominantly from the Ins(1,4,5)P3-sensitive Ca2+ pool, and in this respect thapsigargin was more specific than tBuBHQ. The total increase in [Ca2+] obtained with thapsigargin and Ins(1,4,5)P3 was, on the average, only 7% greater than that with Ins(1,4,5)P3 alone. In contrast, the total increase in [Ca2+] obtained with tBuBHQ and Ins(1,4,5)P3 was 33% greater than that obtained with only InsP3 (P < 0.05). Although Ca2+ was rapidly mobilized by thapsigargin and tBuBHQ, complete depletion of the Ins(1,4,5)P3-sensitive Ca2+ pool was difficult to achieve. After the release by thapsigargin or tBuBHQ, Ins(1,4,5)P3 induced additional Ca2+ release. The additional Ins(1,4,5)P3-induced Ca2+ release was not altered by supramaximal concentrations of thapsigargin and tBuBHQ, or by Bafilomycin A1, an inhibitor of V-type ATPases, but was decreased by prolonged treatment with the ER Ca(2+)-ATPase inhibitors. These results suggest the existence of distinct uptake and release compartments within the Ins(1,4,5)P3-sensitive Ca2+ pool. When treated with the inhibitors, the two compartments became distinguishable on the basis of their Ca2+ permeability. Apparently, thapsigargin and tBuBHQ readily mobilized Ca2+ from the uptake compartment, whereas Ca2+ from the release compartment could be mobilized only very slowly, in the absence of Ins(1,4,5)P3.
Digestion of damaged DNA by the T7 DNA polymerase-exonuclease
Authors: Koehler D R , Hanawalt P C
Published: August 2015

We have investigated the 3′-5′-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4′-hydroxymethyl-4,5′,8-trimethylpsoralen and 5′-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.
Cross-talk between muscarinic- and adenosine-receptor signalling in the regulation of cytosolic free Ca2+ and insulin secretion
Authors: Biden T J , Browne C L
Published: August 2015

The effects of A1-adenosine-receptor occupation on Ca2+ handling in the insulin-secreting RINm5F cell line were investigated. The selective A1-agonist N6-cyclopentyladenosine (CPA) had no effect itself on the cytosolic free Ca2+ concentration in cells loaded with Fura 2. However, CPA (1) attenuated the rise due to activation of voltage-gated Ca2+ channels with Bay K 8644, and (2) caused a secondary increase (EC50 approx. 300 nM) if added after the primary Ca(2+)-mobilizing agonists vasopressin or carbamoylcholine (carbachol). Prior addition of CPA (10 microM) also potentiated (by approx. 20%) the subsequent Ca2+ peak due to maximal (100 microM) carbachol, but did not alter the EC50 of the carbachol response. Detailed analysis of the secondary rise in Ca2+ revealed further features. First, it was due to mobilization from intracellular stores, since it persisted in the absence of extracellular Ca2+. Second, it was associated with a rapid (5-15 s) increase in phospholipase C (PLC) activity, as measured by h.p.l.c. analysis of Ins(1,4,5)P3. This increase was only apparent after prior stimulation with carbachol. Third, and unlike the response to carbachol, it was mediated by a pertussis-toxin-sensitive G-protein. Fourth, it was not secondary to a decrease in cyclic AMP. Fifth, it was absolutely dependent on continued occupation of the primary receptor, since it was abolished if carbachol was displaced with the antagonist atropine. This implies a dynamic cross-talk between the two receptor coupling systems, rather than covalent modification as a result of the prior activation of PLC. Sixth, it was not associated with any desensitization of the ability of CPA to inhibit forskolin-stimulated adenylate cyclase activity. Glyceraldehyde (10 mM)-induced insulin secretion was also potently inhibited by CPA > 10 nM, but the secretory response to 100 microM carbachol was unaffected up to 10 microM. The results suggest that, in vivo, adenosine would inhibit secretion due to carbohydrate nutrients much more effectively than that due to stimuli which activate PLC.
Cloning of the gene for glutamate decarboxylase and its expression during conidiation in Neurospora crassa
Authors: Hao R , Schmit J C
Published: August 2015

Neurospora crassa glutamate decarboxylase (GAD) is produced during conidiation and stored in dormant conidia. Polyclonal antibody was generated to GAD that had been purified to homogeneity. The anti-GAD antibody was specific for N. crassa GAD and inhibited GAD activity. The level of GAD protein decreased during conidial germination, indicating that GAD was degraded during this phase of development. The anti-GAD antibody was used to isolate a cDNA clone of GAD from a lambda ZAP cDNA expression library. Escherichia coli containing a plasmid with the cDNA insert produced GAD activity. The cDNA clone contained a 2.6 kbp insert and hybridized to a 2.6 kb mRNA species from conidiating cultures of N. crassa. GAD mRNA was not present in vegetative hyphae. In conidiating cultures, GAD mRNA was first detected when conidia began to appear. The level of GAD mRNA increased as conidiation progressed. This is the first example of the cloning of an enzyme that is regulated at the level of mRNA during the asexual developmental cycle of N. crassa.
Sex-dependent expression and growth hormone regulation of class alpha and class mu glutathione S-transferase mRNAs in adult rat liver
Authors: Srivastava P K , Waxman D J
Published: August 2015

The sex-dependent expression and growth hormone (GH) regulation of rat liver glutathione S-transferase (GST) was examined using oligonucleotide probes that distinguish between closely related class Alpha (Ya1, Ya2, Yc) and class Mu (Yb1, Yb2, Yb3) GST mRNAs [Waxman, Sundseth, Srivastava and Lapenson (1992) Cancer Res. 52, 5797-5802]. Northern-blot analysis revealed that the steady-state levels of GST Ya1, Yb1 and Yb2 mRNAs are 2.5-3-fold higher in male as compared with female rat liver. In contrast, GST Yc and Ya2 mRNAs were expressed at a 2-3-fold higher level in female rat liver. Microsomal GST mRNA did not exhibit significant sex-dependent differences in rat liver. Treatment of male rats with GH by continuous infusion suppressed expression of the male-dominant GST Ya1, Yb1 and Yb2 mRNAs to levels at or below those found in female rat liver. This suppressive effect of GH was liver-specific, insofar as GH treatment did not alter kidney GST Ya1 mRNA levels. Hypophysectomy increased expression of the male-dominant GSTs, particularly in female rats (e.g. 8-fold elevation of GST Ya1 mRNA). GST Yc mRNA was increased approx. 2-fold in hypophysectomized males, indicating that this mRNA is subject to negative regulation by one or more pituitary-dependent factors. Continuous GH treatment of the hypophysectomized rats suppressed the expression of mRNA of GSTs Ya1, Yb1 and Yb2 when given as a continuous infusion, but not when given by an intermittent (twice daily) GH-injection schedule. Combination of continuous exposure to GH with thyroxine treatment resulted in a more complete suppression of GSTs Ya1, Yb1 and Yb2. In contrast, thyroxine increased the expression of GST Yc in hypophysectomized rats. These studies establish that several Alpha and Mu class GSTs are expressed in a sex-dependent fashion in adult rat liver, where they are regulated by multiple pituitary-dependent hormones through pretranslational mechanisms.
Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria
Authors: Fiermonte G , Walker J E , Palmieri F
Published: August 2015

The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been expressed at an abundant level in Escherichia coli. It accumulates in the bacterium as inclusion bodies, and none of the protein was detected in the bacterial inner membrane. The mitochondrial ADP/ATP carrier, a member of the same super-family of transport proteins as the oxoglutarate carrier, has also been expressed in E. coli. However, the expression of the ADP/ATP carrier in bacteria retards their growth, and so the levels of expression that were attained were lower than those of the oxoglutarate carrier. The oxoglutarate carrier inclusion bodies have been disaggregated with the detergent N-dodecanoyl-sarcosine, and the protein has been incorporated into liposomes. In its ability to transport oxoglutarate and malate and other known substrates of the carrier in mitochondria, and in its inhibition characteristics by a wide range of non-competitive and competitive inhibitors, this reconstituted oxoglutarate carrier is similar to the natural protein in the inner membranes of mitochondria, and to the carrier that has been purified from mitochondria and reconstituted in liposomes. These experiments remove significant obstacles to crystallization trials and to site-directed mutagenesis of the oxoglutarate carrier.
Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript
Authors: Ross V L , Board P G
Published: August 2015

Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library. Both clones are incomplete and appear to result from alternative splicing. One clone is missing the sequence encoding exon 4 and the other is missing exon 8. The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones. This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process. By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli. In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.
Exogenous substrate stimulates autodephosphorylation of cyclic-AMP-dependent protein kinase II
Authors: Gjertsen B T , Fauske B , Døskeland S O
Published: August 2015

The autophosphorylated regulatory subunit (32P-RII) of cyclic-AMP-dependent protein kinase II was efficiently dephosphorylated by its C subunit in the absence of added ADP, provided that Mg/ATP and a standard protein kinase peptide substrate were present. This raises the possibility that autodephosphorylation could be significant in the intact cell. Only the cyclic-AMP-complexed free form of 32P-RII was efficiently dephosphorylated, indicating that the autodephosphorylation was intermolecular. Autodephosphorylation of 32P-RII in the presence of MgATP and kemptide occurred with formation of [gamma-32P]ATP, suggesting transfer of 32P of phospho-RII to a transient C*(MgADP) complex formed during the forward kinase reaction with peptide as substrate. Autodephosphorylation promoted by phosphorylation of exogenous substrates could operate also for other kinases conforming to a mechanism where MgADP remains bound to the active site after the other product (phosphorylated substrate) has left the catalytic complex.
Ascorbic acid accumulation and transport in human fibroblasts
Authors: Welch R W , Bergsten P , Butler J D , Levine M
Published: August 2015

As the initial step in the use of fibroblasts as a model system for ‘in situ kinetics’, ascorbic acid (vitamin C) accumulation in normal human fibroblasts was investigated for the first time. Ascorbic acid was transported into fibroblasts and accumulated against a concentration gradient up to 20-fold, as measured by h.p.l.c. with coulometric electrochemical detection. Ascorbic acid accumulation was mediated by two concentration-dependent transport activities. The first was a high-affinity activity with an apparent Km of 6 microM and an apparent Vmax. of 203 microM/h, and the second was a low-affinity activity with an apparent Km of 5 mM and an apparent Vmax. of 1.8 mM/h. Both activities were inhibited by metabolic inhibitors and inhibitors of ascorbic acid transport in human neutrophils. The low-affinity transporter could not be accounted for by diffusion. Although the high-affinity transport activity was comparable with that described for human neutrophils, the low-affinity transporter was different. These data provide the first evidence that two-component ascorbic acid transport may be a generalized mechanism for accumulation of this vitamin in humans.
Species and tissue distribution of the regulatory protein of glucokinase
Authors: Vandercammen A , Van Schaftingen E
Published: August 2015

Rat liver is known to contain a regulatory protein that inhibits glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver glucokinase. Rat, mouse, rabbit, guinea-pig and pig liver, all of which contain glucokinase, also contained between 60 and 200 units/g of tissue of a regulatory protein displaying the properties mentioned above. By contrast, this protein could not be detected in cat, goat, chicken or trout liver, or in rat brain, heart, skeletal muscle, kidney and spleen, all tissues from which glucokinase is missing. Fructose 1-phosphate stimulated glucokinase in extracts of human liver, indicating the presence of regulatory protein. In addition, antibodies raised against rat regulatory protein allowed the detection of an approximately 60 kDa polypeptide in rat, guinea pig, rabbit and human liver. The livers of the toad Bufo marinus, of Xenopus laevis and of the turtle Pseudemys scripta elegans contained a regulatory protein similar to that of the rat, with, however, the major difference that it was not sensitive to fructose 6-phosphate or fructose 1-phosphate. In rat liver, the regulatory protein was detectable 4 days before birth. Its concentration increased afterwards to reach the adult level at day 30 of extrauterine life, whereas glucokinase only appeared after day 15. In the liver of the adult rat, starvation and streptozotocin-diabetes caused a 50-60% decrease in the concentration of regulatory protein after 7 days, whereas glucokinase activity fell to about 20% of its initial level. When 4-day-starved rats were refed, or when diabetic rats were treated with insulin, the concentration of regulatory protein slowly increased to reach about 85% of the control level after 3 days, whereas the glucokinase activity was normalized after the same delay. The fact that there appears to be no situation in which glucokinase is expressed without regulatory protein is in agreement with the notion that the regulatory protein forms a functional entity with this enzyme.
Transcriptional modulation of cartilage-specific collagen gene expression by interferon γ and tumour necrosis factor α in cultured human chondrocytes
Authors: Reginato A M , Sanz-Rodriguez C , Diaz A , Dharmavaram R M , Jimenez S A
Published: August 2015

To examine the possibility that cytokines produced in inflamed joint tissues may contribute to the loss of articular cartilage by causing inhibition of synthesis of cartilage-specific matrix macromolecules, we studied the effects of interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha), alone and in combination, on the expression of the genes for types-II, -IX and -XI collagens in cultured human chondrocytes. Chondrocytes isolated from human fetal epiphyseal cartilage by sequential enzymic digestions were cultured in the presence of IFN gamma (30 pM), TNF alpha (15 pM) or a combination of suboptimal concentrations of both cytokines (1.5 pM IFN gamma plus 0.3 pM TNF alpha). IFN gamma caused a maximal decrease of 23.3-32.6% in the biosynthesis of collagen by chondrocytes. TNF alpha was a more potent inhibitor causing a 42.8-45.3% decrease at one-half the concentration of IFN gamma. A synergistic inhibitory effect of 58.2% was observed with the combination of 1.5 pM IFN gamma plus 0.3 pM TNF alpha. Electrophoretic analysis of the biosynthesized proteins showed a co-ordinate decrease in the production of the three cartilage-specific collagen types II, IX and XI. These effects were accompanied by parallel changes in the steady-state levels of their corresponding mRNAs. In vitro transcription assays showed that the collagen inhibitory effects of the cytokines occurred largely at the transcriptional level. Similar effects of the cytokines were observed on biosynthesis of types-II, -IX and -XI collagens and steady-state mRNA levels for type-II collagen by chondrocytes obtained from adult articular cartilage. These observations indicate that IFN gamma and TNF alpha can induce a synergistic inhibition of the synthesis of cartilage-specific collagens by fetal and adult human chondrocytes and suggest that these effects may contribute to the articular cartilage loss that occurs in inflammatory joint diseases.
Purification, characterization and modulation of a microsomal carboxylesterase in rat liver for the hydrolysis of acyl-CoA
Authors: Mukherjee J J , Jay F T , Choy P C
Published: August 2015

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.
Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins
Authors: Zocchi E , Guida L , Franco L , Silvestro L , Guerrini M , Benatti U , De Flora A
Published: August 2015

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.
Physiological modulation of the uptake and fate of glucose in brown adipose tissue
Authors: Sugden M C , Holness M J
Published: August 2015

Glucose utilization indices (GUI values) and rates of fatty acid synthesis in interscapular brown adipose tissue (IBAT) varied during the diurnal cycle in virgin and late-pregnant rats permitted unrestricted access to food. In virgin rats, peak GUI values and lipogenic rates were observed at the end of the dark (feeding) phase, but were not sustained during the light phase. Whereas peak GUI values were comparable with those observed during re-feeding after 24 h starvation, maximum rates of IBAT fatty acid synthesis in virgin rats during the diurnal cycle were only approx. 25% of those measured during re-feeding after 24 h starvation. Despite hyperphagia, GUI values during the diurnal cycle in late-pregnant rats fed ad libitum were generally lower than those of age-matched virgin controls. The percentage of pyruvate dehydrogenase complex present in the active form (PDHa) was also significantly decreased. Suppression of GUI and PDHa was not parallelled by suppression of fatty acid synthesis. IBAT GUI values in late-pregnant rats during chow re-feeding ad libitum after 24 h starvation were only 25% of those of corresponding virgin controls, and stimulation of fatty acid synthesis was also dramatically attenuated. The suppression of IBAT GUI values after re-feeding in pregnancy was not due to depletion of GLUT 4 protein. The results are discussed in relation to the importance of glucose as a precursor for fatty acid synthesis in IBAT.
Lactase-phlorizin hydrolase and aminopeptidase N are differentially regulated in the small intestine of the pig
Authors: Torp N , Rossi M , Troelsen J T , Olsen J , Danielsen E M
Published: August 2015

The longitudinal expression of two brush-border enzymes, lactase-phlorizin hydrolase (EC 3.2.1.23/62) and aminopeptidase N (EC 3.4.11.2), was studied in the small intestine of the post-weaned pig. Whereas the level of mRNA, encoding aminopeptidase N (relative to that of beta-actin), only varied moderately from the duodenum to the terminal ileum, the amount of lactase-phlorizin hydrolase mRNA exhibited a sharp maximum in the proximal jejunum. For both enzymes, the level of protein synthesis, studied in cultured mucosal explants, correlated well with the level of mRNA, and no major variation in post-translational processing or intracellular transport was observed along the intestine. The mRNA/specific-activity ratio for both enzymes was markedly (3-5-fold) higher in the duodenum and proximal jejunum, compared with the ileum. This indicates an increased proximal turnover rate, most likely caused by the presence in the gut lumen of pancreatic proteases. In neonatal animals, the level of mRNA for lactase-phlorizin hydrolase in both proximal and distal regions of the intestine was of the same magnitude as in the proximal jejunum of the post-weaned pigs. Our results point to two mechanisms that affect the expression of lactase-phlorizin hydrolase in the pig during development: (1) a primary regulation at the level of mRNA (predominantly in the ileum); (2) an increased rate of turnover of the enzyme, mainly in the duodenum and proximal jejunum, and most likely due to an increased secretion into the gut lumen of pancreatic proteases (a mechanism also affecting aminopeptidase N and probably other brush-border enzymes as well).
Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene
Authors: Bingle C D , Gitlin J D
Published: August 2015

To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 ‘forkhead’ family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm.
The helix-loop-helix transcription factor USF (upstream stimulating factor) binds to a regulatory sequence of the human insulin gene enhancer
Authors: Read M L , Clark A R , Docherty K
Published: August 2015

Two important sequence elements, designated insulin enhancer binding site 1 (IEB1) or NIR and IEB2 or FAR, are involved in regulating expression of the rat insulin I gene. These elements bind a helix-loop-helix transcription factor, insulin enhancer factor 1 (IEF1). The IEB1 site is highly conserved among insulin genes but the IEB2 site is not conserved. To investigate the factors binding at the equivalent IEB1 and IEB2 sites in the human insulin gene enhancer, electrophoretic mobility shift assays were performed using a variety of cell extracts and probes specific for the homologous IEB1 and IEB2 sites. The results indicate that a factor with similar tissue distribution and binding characteristics to those of IEF1 binds to the IEB1 site in the human insulin gene, but that a separate factor, identified as the adenovirus major late transcription factor [MLTF, or upstream stimulating factor (USF)] binds to the IEB2 site.
Induction of pterin synthesis is not required for cytokine-stimulated tryptophan metabolism
Authors: Sakai N , Saito K , Kaufman S , Heyes M P , Milstien S
Published: August 2015

Activation of the immune system which occurs in inflammatory disease leads to parallel increases in pterin synthesis and increased production of neuroactive L-tryptophan metabolites. Several model systems were studied to determine whether pterins, which are cofactors for hydroxylation reactions, could be required in the oxidative kynurenine pathway of L-tryptophan degradation. Treatment of mice with interferon-gamma increased L-tryptophan metabolism without any corresponding change in tissue biopterin concentrations. Cytokine-treated human fibroblasts, macrophages and glioblastoma cells all showed increases in kynurenine production, which were completely independent of pterin synthesis. When pterin synthesis de novo was blocked, either by an inhibitor of GTP cyclohydrolase or because of a genetic deficiency of one of the enzymes of the pathway of pterin biosynthesis, cytokine-stimulated increases in tryptophan metabolism were unaffected. Furthermore, increasing intracellular tetrahydrobiopterin concentrations by treating cells with sepia-pterin also had no effect on markers of tryptophan metabolism. Therefore, both normal and cytokine-stimulated L-tryptophan metabolism appears to be completely independent of pterin biosynthesis.
Characterization of a complement-fragment-C5a-stimulated calcium-influx mechanism in U937 monocytic cells
Authors: Monk P N , Partridge L J
Published: August 2015

The mechanism by which complement fragment C5a elevates intracellular Ca2+ ([Ca2+]i) levels in two cell types, a monocytic cell line, U937, and neutrophils, has been investigated by the use of fluorometric and radiometric techniques. In U937 cells the influx of extracellular Ca2+ can be distinguished from the release of intracellular Ca2+ stores in terms of dose-responsiveness to C5a and sensitivity to pertussis-toxin poisoning. This suggests that the mechanism of Ca2+ influx in these cells is at least partially independent of both the production of inositol phosphates and elevation of internal Ca2+ concentration. The C5a-stimulated influx of 45Ca2+ into U937 cells is inhibited by a series of metal ions (Zn2+ > Co2+ > Mn2+ > Sr2+ approximately equal to Ni2+ > La3+). The stimulated influx of Ca2+ into neutrophils is inhibited differently (Ni2 >> Co2+ > Zn2+ approximately equal to La3+ > Mn2+ approximately equal to Sr2+), is less sensitive to C5a and both the influx of extracellular Ca2+ and the release of intracellular stores are equally sensitive to pertussis toxin treatment. Taken together these results indicate that [Ca2+]i is controlled in U937 monocytes by mechanisms distinct from those which appear to operate in other myeloid cells, such as neutrophils, stimulated with C5a and formylpeptide.
Met-8 of the β1-bungarotoxin phospholipase A2 subunit is essential for the phospholipase A2-independent neurotoxic effect
Authors: Chu S T , Chu C C , Tseng C C , Chen Y H
Published: August 2015

beta 1-Bungarotoxin consists of a phospholipase A2 subunit and a non-phospholipase A2 subunit. The toxin was oxidized with a 100-fold molar excess of chloramine T with respect to the methionine content of the protein in 0.1 M Tris/HCl at pH 8.5 and at room temperature. Reactivities of the two methionine (Met-6 and Met-8 of the phospholipase A2 subunit), five histidine, 14 tyrosine and one tryptophan residues of one toxin molecule with chloramine T were assessed from the change in intrinsic fluorescence and amino acid composition of the protein. Met-8 and one tyrosine on the phospholipase A2 subunit and less than one histidine were oxidized, while Met-6 remained intact after 30 min of reaction. One histidine and approx. two tyrosine residues were oxidized when both methionine residues were oxidized after 90 min of reaction. The sole tryptophan was oxidized slightly throughout the reaction. The chloramine T oxidation did not destroy the two Ca(2+)-binding domains, though it modified the toxin to become less effective at binding Ca2+. The modified toxin obtained after 30 or 90 min reaction time retained 65% or 40% of the phospholipase A2 activity of the parent toxin, but both were not lethal to mice and showed a very weak ability to induce the indirectly evoked contraction of chick biventer cervicis muscle. It is suggested that Met-8 may play an important role in the phospholipase A2-independent interaction with the nerve terminal membrane during the neurotoxic effect of beta 1-bungarotoxin.
Evidence for allosteric regulation of succinyl-CoA synthetase
Authors: Um H D , Klein C
Published: August 2015

We have previously reported that distinctly different concentrations of GDP stimulate the phosphorylation and dephosphorylation of p36, the alpha-subunit of succinyl-CoA synthetase (SCS) in Dictyostelium discoideum. In this present study, we have investigated the mechanism underlying these dual effects of GDP. Dephosphorylation of p36 is induced by relatively high levels of GDP and is coincident with the formation of GTP. This indicates that, at high concentrations, GDP serves as a substrate of SCS. However, 100-fold lower concentrations of GDP, which do not bind to the catalytic site to induce SCS dephosphorylation, stimulate p36 phosphorylation. This stimulation is not diminished by dilution of the sample, and is retained during purification of the protein. Gel-filtration analyses indicate that SCS in our system behaves as a non-interacting alpha beta dimer, the hydrodynamic behaviour of which is not altered by the presence of added GDP. The data indicate that altered protein-protein interactions do not account for the stimulation of p36 phosphorylation by low GDP concentrations. We propose that GDP functions as an allosteric regulator of SCS, and experiments using guanosine 5′-[beta-thio]diphosphate (GDP[S]) are shown to distinguish further the allosteric and catalytic binding sites.
Specific inhibition of the activity of the urokinase receptor-mediated cell-surface plasminogen activation system by suramin
Authors: Ellis V , Danø K
Published: August 2015

Urokinase-type plasminogen activator (uPA) is involved in generating the proteolytic activity necessary for invasive processes, and is dependent on binding to its specific cellular receptor (uPAR) for efficient function. We report here that the polysulphonated napthylurea compound suramin inhibits the activity of this cell-associated proteolytic system, in a manner independent of its antagonism of the uPA-uPAR interaction [Behrendt, Rønne and Danø (1993) J. Biol. Chem. 268, 5985-5989], occurring at a 25-100-fold-lower suramin concentration. This inhibition was found to be due to effects on the activation of both pro-uPA and plasminogen. Suramin inhibited plasmin activation of pro-uPA by a non-competitive mechanism (Ki approx. 2 micrograms/ml), which did not involve a direct effect on plasmin catalytic activity. Similarly, its effect on plasminogen activation was not due to a direct inhibition of uPA. The inhibition of plasminogen activation, which occurred exclusively with receptor-bound uPA, appeared to be due to a reversal of the favourable kinetics which result from the activation of cell-associated plasminogen, although suramin did not inhibit the cellular binding of 125I-labelled plasminogen. This suggests that this effect is due to interference with interactions between components of this system on the cell surface, and that suramin may be useful in gaining further insight into the molecular mechanisms involved in the functional assembly of this proteolytic system. Furthermore the effective inhibition of this system by suramin indicates an anti-invasive potential that may contribute to the anti-tumour effect of suramin in vivo.
The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues
Authors: Shimomura O , Kishi Y , Inouye S
Published: August 2015

The regeneration of an active semi-synthetic aequorin, from apoaequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2+ in the cells. The relative rates of the regeneration of semi-synthetic aequorins from apoaequorin and 28 synthetic coelenterazine analogues were compared. The results indicated that the rate is strongly influenced by the analogues used. The regeneration of ordinary aequorin with normal coelenterazine was relatively fast (50% regeneration in 22 min), whereas the rates of regenerating semi-synthetic aequorins with coelenterazine analogues varied widely, and all were slower than that of regenerating ordinary aequorin, except for e-type coelenterazines (containing an extra ethano group). The regeneration with e-type coelenterazines was significantly faster, indicating the possible superiority of e-type analogues in the intracellular regeneration of aequorin, especially when an increased sensitivity to Ca2+ is needed.
Primary structure and characterization of an androgen-stimulated autoantigen purified from mouse seminal-vesicle secretion
Authors: Yu L C , Chen J L , Tsai W B , Chen Y H
Published: August 2015

A protein extract of mouse seminal-vesicle secretion was used to immunize mature mice (Balb/c) of both sexes. Results of Western-blot analyses for these secretory proteins indicated that only one minor protein component could be recognized by the autoantisera prepared from either autoimmunization of male mice or isoimmunization of female mice. The autoantigen was purified from seminal-vesicle secretion. The purified autoantigen retained the ability to induce autoantibody formation. The autoantigen has glycoprotein characteristics: the majority of the carbohydrate is N-linked and the remainder is O-linked. Rabbit antibodies to the autoantigen were used to isolate the corresponding cDNA from a mouse seminal-vesicle cDNA library. The primary structure deduced from the cDNA sequence was confirmed by direct amino acid sequence determination. The results indicate that the core protein consists of 131 amino acid residues. Analysis of the primary structure indicates that the autoantigen has two potential acceptor sites for the N-linked carbohydrate at Asn-12 and Asn-122, three potential phosphorylation sites for casein kinase II at Thr-55, Ser-68 and Thr-76, and three potential phosphorylation sites for protein kinase C at Thr-28, Thr-40 and Thr-124. The core protein and the carbohydrate portion together have a molecular mass of 19 kDa. Results from Western- and Northern-blot analyses for various tissues indicate that the seminal vesicle is the sole organ producing this autoantigen. Expression of this autoantigen gene was stimulated by testosterone.
Defining the roles of the threefold channels in iron uptake, iron oxidation and iron-core formation in ferritin: a study aided by site-directed mutagenesis
Authors: Treffry A , Bauminger E R , Hechel D , Hodson N W , Nowik I , Yewdall S J , Harrison P M
Published: August 2015

This paper aims to define the role of the threefold intersubunit channels in iron uptake and sequestration processes in the iron-storage protein, ferritin. Iron uptake, measured as loss of availability of Fe(II) to ferrozine (due to oxidation), has been studied in recombinant human H-chain ferritins bearing amino acid substitutions in the threefold channels or ferroxidase centres. Similar measurements with recombinant horse L-chain ferritin are compared. It is concluded that significant Fe(II) oxidation occurs only at the H-chain ferroxidase centres and not in the threefold channels, although this route is used by Fe(II) for entry. Investigations by Mössbauer and u.v.-difference spectroscopy show that part of the iron oxidized by H-chain ferritin returns to the threefold channels as Fe(III). This monomeric Fe(III) can be displaced by addition of Tb(III). Fe(III) also moves into the cavity for formation of the iron-core mineral, ferrihydrite. Iron incorporated into ferrihydrite becomes kinetically inert.