bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R1 mRNA 3′-untranslated region cis-trans interaction through a protein kinase C-controlled pathway
Authors: Chen F Y , Amara F M , Wright J A
Published: August 2015

Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3′-untranslated region (3′UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; ‘TPA’) and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3′UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3′UTR.
Solubilization and purification of the retinol-binding protein receptor from human placental membranes
Authors: Sivaprasadarao A , Boudjelal M , Findlay J B C
Published: August 2015

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.
Cholesterol derivative of a new triantennary cluster galactoside directs low- and high-density lipoproteins to the parenchymal liver cell
Authors: Biessen E A L , Vietsch H , Van Berkel T J C
Published: August 2015

We have developed a new triantennary galactoside, in which the terminal galactose moieties are connected to the branching point of the cluster galactoside via a 20 A (2 nm) spacer [TG(20A)]. In vitro binding studies have demonstrated that introduction of a 20 A spacer resulted in avid and specific binding of the triantennary galactoside to the asialoglycoprotein receptor on the parenchymal liver cell. Derivatization of this galactoside with a cholesterol moiety afforded a compound [TG(20A)C] that lowered the serum cholesterol concentration when injected into rats. In the present study we have evaluated the direct effect of TG(20A)C on the in vivo fate of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). A direct association of TG(20A)C with HDL and LDL was observed on mixing these components. Incorporation of TG(20A)C into 125I-HDL and 125I-LDL significantly accelerated the serum decay and concomitantly stimulated the hepatic uptake of these lipoproteins in rats. The liver uptake of TG(20A)C-loaded 125I-HDL or 125I-LDL could be inhibited by 81% and 82% respectively by preinjection of 150 mg of N-acetylgalactosamine, indicating that the enhanced liver uptake proceeded via galactose-specific receptors. More than 96% of the hepatic uptake of TG(20A)C-loaded 125I-HDL could be attributed to the parenchymal cell. Surprisingly, the parenchymal cell also accounted for 93% of the liver association of TG(20A)C-loaded 125I-LDL, suggesting that TG(20A)C stimulates the uptake and processing of both lipoproteins by the asialoglycoprotein receptor on the parenchymal liver cell. This contrasts with earlier data indicating that a triantennary cluster galactoside provided with a 4 A spacer between the terminal galactose moieties and the branching point of the dendrite stimulated hepatic uptake of LDL via the Kupffer cells. The parenchymal cell is the only liver cell type that is capable of irreversibly removing cholesterol from the body in the form of bile acids. The above results imply that administration of TG(20A)C not only facilitates the hepatic uptake of lipoprotein-derived cholesterol (esters) but also their elimination from the body. In addition, it might be possible to utilize TG(20A)C as a targeting device to selectively deliver large drug carriers and possibly genes to the parenchymal liver cell.
The N-ethylmaleimide-sensitive protein thiol groups necessary for sea-urchin egg cortical-granule exocytosis are highly exposed to the medium and are required for triggering by Ca2+
Authors: Whalley T , Sokoloff A
Published: August 2015

It is known that sea-urchin egg cortical-granule exocytosis is inhibited by agents such as N-ethylmaleimide (NEM) which modify thiol groups. The fusion-related proteins modified by these agents have yet to be identified, nor is there information regarding the topography of these thiol groups. Furthermore, the step in cortical-granule exocytosis at which these thiol groups participate is unknown. In this study we have investigated the topological properties of, and the temporal requirement for the function of, the fusion-related thiol groups by treating the isolated exocytotic apparatus with high-molecular-mass dextrans and BSA carrying thiol-reactive 3-(2-pyridyldithio)propionate groups. The dextran derivatives inhibited exocytosis. The BSA derivative was much less inhibitory. Inhibition was reversed by treatment with dithiothreitol. When NEM was added to the dextran-derivative-treated exocytotic apparatus, treatment with dithiothreitol completely reversed inhibition, indicating that the dextran derivatives inhibit by reacting at the NEM-sensitive sites. A pulse of Ca2+ applied in the presence of inhibitors did not trigger any fusion following the removal of the inhibitor by dithiothreitol. These data show that the thiol groups, the modification of which by NEM inhibits exocytosis, are exposed to the medium in terms of their accessibility to macromolecules. They also show that the fusion-related thiol groups are required during the Ca(2+)-dependent stage of exocytosis.
Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme
Authors: Steinkühler C , Carrì M T , Micheli G , Knoepfel L , Weser U , Rotilio G
Published: August 2015

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.
A 5′ splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated proα1(I) chains with a non-collagenous insertion destabilizing the triple helix
Authors: Bateman J F , Chan D , Moeller I , Hannagan M , Cole W G
Published: August 2015

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3′ limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.
Isolation and characterization of two different flavodoxins from the eukaryote Chlorella fusca
Authors: Peleato M L , Ayora S , Inda L A , Gómez-Moreno C
Published: August 2015

Two different molecular forms of flavodoxin from the green alga Chlorella fusca have been purified to homogeneity and their properties compared. The molecular masses are 22 kDa (flavodoxin I) and 20 kDa (flavodoxin II). Western blots of axenic crude extract show the two bands. Both are single polypeptide chains and their N-terminal sequences differ but are very similar. Each form contains 1 mol of FMN/mol of apoprotein, exhibits a typical flavodoxin u.v.-visible absorption spectrum and does not contain covalently bound phosphate. The oxidation-reduction properties of the FMN in the flavodoxins differ considerably. Redox potentials of flavodoxin I at pH8 are -240 mV for the oxidized/semiquinone couple and -350 mV for the semiquinone/hydroquinone couple. Flavodoxin II gives more electronegative values: -278 mV and -458 mV respectively. Flavodoxin II fulfils better the redox requirements for photosynthetic electron transport and, as expected, it is more efficient at mediating NADP+ photoreduction in the photosynthetic electron flow. A new h.p.l.c. method for flavodoxin purification is described, which is useful for the isolation of very similar anionic proteins.
Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli
Authors: Bauminger E R , Treffry A , Hudson A J , Hechel D , Hodson N W , Andrews S C , Levi S , Nowik I , Arosio P , Guest J R , HARRISON P M
Published: August 2015

Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores. Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation. Mössbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin. Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN). A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III). Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex. Mössbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins. Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components. This provides further evidence that the ferritin protein shell can influence the structure of its iron core.
‘CO2-ligated’ cytochrome c oxidase: characterization and comparison with the Cl- -ligated enzyme
Authors: Moody A J , Richardson M , Spencer J P , Brandt U , Rich P R
Published: August 2015

A form of fully oxidized bovine heart cytochrome c oxidase that is induced by CO2/HCO3- is described. The ligand-binding properties of this form are similar to those of Cl(-)-ligated oxidase [Moody, Cooper and Rich (1991) Biochim. Biophys. Acta 1059, 189-207]. Both bind cyanide at a rate (0.2 M-1.s-1 at pH 6.5) intermediate between the rate of binding to the fast and slow forms of the enzyme, and binding of formate to both is almost undetectable. They are also similar in showing poor reactivity with H2O2, or with CO in the presence of O2, which, with fast oxidase, induce the formation of the ‘ferryl’ and ‘peroxy’ states respectively. However, there is a clear difference in the near-u.v./visible absorption spectra of the two forms; that induced by CO2/HCO3- has a Soret maximum at 427 nm whereas Cl(-)-ligated oxidase has a Soret maximum similar to that of fast oxidase at about 424 nm. It appears that both CO2/HCO3- and Cl- are members of a class of ligands that lowers the reactivity of the binuclear centre but does not impede intramolecular electron transfer from haem a to the binuclear centre, unlike the putative endogenous ligand responsible for slow oxidase.
Cultured cerebellar granule cells, but not astrocytes, produce an ester of ganglioside GD1b, presumably GD1b monolactone, from exogenous GD1b
Authors: Bassi R , Riboni L , Tettamanti G
Published: August 2015

Granule cells and astrocytes from rat cerebellum were fed in culture with 2 microM ganglioside [Gal-3H]GD1b and then analysed for the presence of carboxyl esters of that ganglioside. Before extraction and purification of gangliosides, cells were treated with NaBH4 under conditions that would allow complete reductive cleavage of carboxyl ester linkages, [Gal-3H]GD1b monolactone and dilactone being used as reference esters of GD1b. These conditions, established by adding harvested cells (250 micrograms of protein) with 0.01-2 nmol of standard [Gal-3H]GD1b monolactone or dilactone and [Gal-3H]GD1b-1ol or -2ol formed respectively, consisted of an NaBH4/cell protein ratio of 2:1 (w/w). Cerebellar granule cells, but not astrocytes, were able to produce a radioactive compound which was identified as GD1b-1ol. The formation of this compound increased with pulse (up to 4 h) and chase (up to 3 h) time after a 2 h pulse and also occurred when ganglioside endocytosis was blocked. It can be concluded that cerebellar granule cells are able to convert ganglioside GD1b into a carboxyl ester form, presumably GD1b monolactone. The natural occurrence of the same GD1b carboxyl ester in cerebellar granule cells was also demonstrated.
The hemispheric functional expression of the thyrotropin-releasing-hormone receptor is not determined by the receptors' physical distribution
Authors: Matus-Leibovitch N , Nussenzveig D R , Gershengorn M C , Oron Y
Published: August 2015

The thyrotropin-releasing-hormone receptor (TRH-R) is a member of a family of the G-protein-coupled receptors that share structural similarities and exert their physiological action via the inositol lipid signal-transduction pathway. The TRH-R when expressed in Xenopus oocytes exhibits marked preference of the response (increased chloride conductance) for the animal hemisphere. Whereas the rat TRH-R functional distribution was strongly asymmetric (animal/vegetal ratio = 9.5), the mouse TRH-R exhibited a significantly lower ratio (3.9). Truncation of the last 59 amino acids of the C-terminal region of the mouse TRH-R did not lead to any changes in the functional hemispheric distribution. Despite the polarization of response, receptor number was similar on both hemispheres. Moreover, the apparent half-life of the functional expression of the TRH-R was approx. 4 h on both hemispheres when the expression was inhibited by a specific antisense oligonucleotide. Inhibition of total protein synthesis with cycloheximide affected hemispheric responses mediated by each of the three TRH-Rs tested in a qualitatively different way. These results suggest that an additional, rapidly degraded, protein modulates the functional hemispheric expression of the TRH-Rs.
Effect of chronic incubation of CaCo-2 cells with eicosapentaenoic acid (20:5, n-3) and oleic acid (18:1, n-9) on triacylglycerol production
Authors: Ranheim T , Gedde-Dahl A , Rustan A C , Drevon C A
Published: August 2015

CaCo-2 monolayers, cultured for 1 week after reaching confluence, were incubated with micellar solutions of fatty acids for up to 7 days. These conditioned cells were incubated acutely (5 h) with eicosapentaenoic acid and oleic acid, and the levels of cell-associated and secreted triacylglycerol were determined. With acute addition of oleic acid, both cell-associated and secreted triacylglycerol were decreased in cells chronically exposed to eicosapentaenoic acid. This effect was observed after as little as 2 days of chronic incubation with eicosapentaenoic acid. A further decrease was found when these cells were incubated acutely with eicosapentaenoic acid, regardless of which radioisotopes were used to label precursors in the incubation media. The secretion of both labelled and total triacylglycerol and apolipoprotein B was reduced approximately 50% in cells incubated chronically with eicosapentaenoic acid. The amounts of triacylglycerol and apolipoprotein B within the cells were not decreased by chronic exposure to eicosapentaenoic acid. Our data indicate that CaCo-2 cells chronically incubated with eicosapentaenoic acid secrete significantly less triacylglycerol than cells incubated chronically with oleic acid. When eicosapentaenoic acid was also included acutely, triacylglycerol secretion was reduced even more. We conclude that chronic exposure of eicosapentaenoic acid to this intestinal cell type reduces the rate of chylomicron secretion and may help explain the decreased postprandial lipaemia observed in humans taking fish oil supplements.
Identification of one- and two-chain forms of trypsinogen 1 produced by a human gastric adenocarcinoma cell line
Authors: Koshikawa N , Yasumitsu H , Nagashima Y , Umeda M , Miyazaki K
Published: August 2015

It has previously been reported that two kinds of human gastric adenocarcinoma cell lines (STKM-1 and MKN28) secrete a trypsin-like enzyme. In this study, four molecular forms of the enzyme (26, 25, 24 and 23 kDa on non-reducing SDS/PAGE) were purified from the serum-free conditioned medium of STKM-1 cells. Analysis of N-terminal amino acid sequences showed that the 26 kDa protein was a two-chain form of trypsinogen 1 which had been produced by proteolytic cleavage of the Arg107-Val108 bond of trypsinogen 1, and the 24 kDa protein was the one-chain form of trypsinogen 1. The 25 and 23 kDa proteins were the activated forms of the two-chain and one-chain trypsinogen 1 respectively. Isoelectric focusing gave pI values of 6.3 and 6.6 for the 26 kDa two-chain form and the 24 kDa one-chain form of trypsinogen 1 respectively. Comparison of the proteolytic activities indicated that the one-chain trypsin 1 had amidolytic activity about four times higher than that of the two-chain enzyme.
Inactivation precedes changes in allosteric properties and conformation of d-glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase during denaturation by guanidinium chloride
Authors: Jiang R F , Tsou C L
Published: August 2015

It has been shown that inactivation of several enzymes precedes overall conformational changes of the enzyme molecules as a whole during denaturation [Tsou (1993) Science, 262, 380-381]. However, the relation between inactivation, loss of allosteric properties of oligomeric enzymes and unfolding of the enzyme molecule during denaturation remain little explored. These have now been compared for D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphatase (FruP2ase) during denaturation by guanidinium chloride (GdmCl). GAPDH is completely inactivated at 0.3 M GdmCl but at this GdmCl concentration it still binds NAD+ with negative co-operativity. At 0.4 M GdmCl, inactivation of FruP2ase reaches completion whereas its allosteric properties, including the heterotropic effect of AMP inhibition and K+ activation with positive co-operativity, are only partially affected. Much higher GdmCl concentrations are required to bring about unfolding of the overall structures of both enzymes.
Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A
Authors: Rempel A , Bannasch P , Mayer D
Published: August 2015

Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.
Cloning and initial characterization of the promoter region of the rat cysteine-rich intestinal protein gene
Authors: Levenson C W , Shay N F , Cousins R J
Published: August 2015

Cysteine-rich intestinal protein (CRIP) is an intestinal Zn(2+)-binding protein containing a single copy of the double Zn(2+)-finger arrangement known as the LIM motif. CRIP is developmentally regulated and can be induced by glucocorticoid hormones during the early suckling period. In this report we show that CRIP mRNA levels are induced by dexamethasone in cultured rat intestinal epithelial cells (IEC-6). Analysis of the 2644 bp of the 5′-flanking region of the CRIP gene revealed that the CRIP promoter lacks classical CAAT and TATA boxes but contains GC-rich regions in the proximal end of the promoter that probably function in transcription initiation. In addition to binding sites for transcription factors such as Sp-1, AP-2, OCT and GATA-2, there are multiple glucocorticoid-response elements. CRIP promoter constructs fused to the chloramphenicol acetyltransferase reporter gene and transfected into IEC-6 cells confirmed glucocorticoid responsiveness and the presence of negative acting elements. Mobility-shift assays revealed the presence of nuclear factors that bind to the CRIP promoter as a result of dexamethasone treatment. These experiments provide the initial data required to explore further the regulation of this tissue-specific developmentally regulated Zn(2+)-finger protein.
Characterization of platelet-activating factor binding to human airway epithelial cells: modulation by fatty acids and ion-channel blockers
Authors: Kang J X , Man S F P , Hirsh A J , Clandinin M T
Published: August 2015

Radioligand-binding studies were performed in primary cultured human airway epithelial cells with [3H]PAF to determine whether these cells express platelet-activating factor (PAF) receptors. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites with Kd 1.8 +/- 0.2 nM and Bmax. 21.0 +/- 2.1 fmol/10(6) cells (13,000 receptors/cell). PAF binding increased the intracellular free Ca2+ concentration ([Ca2+]i), indicating functional PAF receptors. Palmitate (C16:0), linoleic acid (C18:2 omega 6) or eicosapentaenoic acid (C20:5 omega 3) was incubated with the cells to test the effect on PAF binding. Incorporation of each fatty acid into cellular phospholipid occurred. [3H]PAF (1 nM) binding decreased in cells supplemented with C20:5 omega 3, but increased in the cells supplemented with C16:0. Scatchard analysis revealed that the inhibition of PAF binding by supplementation with C20:5 omega 3 was due to a decrease in both affinity and number of PAF receptors. PAF-stimulated increase in [Ca2+]i was also decreased by 60% in cells supplemented with C20:5 omega 3. Verapamil, a Ca(2+)-channel blocker, and amiloride, a Na(+)-channel blocker, inhibited specific binding of [3H]PAF to the cells, with IC50 4-5 microM and 0.2 mM respectively. Diphenylamine-2-carboxylate (DPC), a Cl(-)-channel blocker, dramatically increased PAF binding to the cell in a dose-dependent manner. Scatchard analysis revealed that verapamil and amiloride decreased both binding affinity and number of PAF receptors, whereas DPC increased PAF binding sites without affecting binding affinity. These results demonstrate that human airway epithelial cells have a functional receptor for PAF and that PAF receptor binding can be modulated by exogenous fatty acids and by ion-channel blockers.
Different zonal distribution of the asialoglycoprotein receptor, the α2-macroglobulin receptor/low-density-lipoprotein receptor-related protein and the lipoprotein-remnant receptor of rat liver parenchymal cells
Authors: Voorschuur A H , Kuiper J , Neelissen J A M , Boers W , Van Berkel T J C
Published: August 2015

Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.
Modulation of complement regulatory function and measles virus receptor function by the serine-threonine-rich domains of membrane cofactor protein (CD46)
Authors: Iwata K , Seya T , Ueda S , Ariga H , Nagasawa S
Published: August 2015

Three major membrane cofactor protein (MCP) phenotypes with different serine-threonine (ST)-rich regions, namely STc (L-phenotype), STBC (H or U phenotype) and STABC, and the MCP without the ST domain (delta ST) were expressed in Chinese hamster ovary (CHO) cells by transfecting the respective cDNAs. The expressed molecules migrated with a larger molecular mass on SDS/PAGE than those expected from their amino acid sequences. O-Glycanase digestion showed that this was due to O-linked sugar chains. The apparent sugar contents in each ST segment were compatible with their serine and threonine contents in the ST regions. The functional properties of these phenotypes as inhibitors of human complement (C) and receptors of measles virus (MV) were compared. The classical pathway-dependent CHO cell lysis by human C was more effectively suppressed by the expressed delta ST and STC than by the STABC and STBC phenotypes, although the difference was not so prominent. In contrast, alternative C pathway-dependent CHO-cell lysis was most effectively suppressed by the STABC phenotype and was only slightly blocked by the ST-deleted mutant. MV infection occurred with all of the phenotypes, but the infectious dose required to cause the same level of syncytium formation was 100-times higher in large ST (STABC and STBC) than in small ST (STC and delta ST) phenotypes. Thus, the ST domain serves as a functional modulator in MCP: MCP with a large ST domain having high O-linked sugar contents is favourable to the effective suppression of both the alternative C pathway-mediated cytolysis and MV infection, whereas MCP with a small ST domain is favourable to the suppression of the classical C pathway.
Dexamethasone differentially affects interleukin 1β- and cyclic AMP-induced nitric oxide synthase mRNA expression in renal mesangial cells
Authors: Kunz D , Walker G , Pfeilschifter J
Published: August 2015

Inducible nitric oxide synthase (iNOS) is expressed in renal mesangial cells in response to two principal classes of activating signals that interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)-1 beta or tumour necrosis factor alpha and agents that elevate cellular levels of cyclic AMP (cAMP). We examined whether dexamethasone differentially affects iNOS induction in response to IL-1 beta and a membrane-permeable cAMP analogue, N6,O-2′-dibutyryladenosine 3′,5′-phosphate (Bt2cAMP). Nanomolar concentrations of dexamethasone suppress IL-1 beta- as well as Bt2cAMP-induced iNOS protein expression and production of nitrite, the stable end product of nitric oxide (NO) formation. In contrast, dexamethasone prevents induction of iNOS mRNA in response to Bt2cAMP without affecting IL-1 beta-triggered increase in iNOS mRNA levels. These data suggest that dexamethasone acts at different levels, depending on the stimulus used to suppress iNOS induction in mesangial cells.
Effect of unesterified cholesterol on the activity of cholesteryl ester transfer protein
Authors: Rajaram O V , Chan R Y S , Sawyer W H
Published: August 2015

Cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from high-density lipoprotein to triacylglycerol-rich lipoproteins and the transfer of triacylglycerols in the reverse direction. The activity of CETP has been studied using a continuous fluorescence assay which measures the excimer fluorescence of cholesteryl 1-pyrene decanoate in a synthetic donor microemulsion as the indicator of cholesteryl ester transfer. Emulsions were composed of cholesteryl oleate and egg phosphatidylcholine and had an average particle size of 14 +/- 1 nm as calculated from the molar volume of the components. The effect of changing the physical state of the emulsion surface was examined by including unesterified cholesterol in the donor and acceptor particles. The rate of CETP-induced transfer of the fluorescent cholesteryl ester between microemulsion particles increased when unesterified cholesterol was present at concentrations up to 17 mol% relative to phospholipid. The presence of cholesterol also changed the exchange kinetics from an apparent single-exponential to a double-exponential phenomenon. Binding of CETP to the emulsion surface was accompanied by an enhancement of fluorescence which was used to measure the binding equilibria. The enhancement of exchange due to the presence of cholesterol did not correlate with any increased binding of CETP to the emulsion surface. The presence of unesterified cholesterol in the donor did not affect the rate of transfer of the fluorescent cholesteryl ester when unlabelled emulsion was replaced by high-density lipoprotein as the acceptor. The studies demonstrate the use of microemulsions of defined size and composition for the study of the mechanism of action of CETP.
Glycoinositol-phospholipid profiles of four serotypically distinct Old World Leishmania strains
Authors: Schneider P , Schnur L F , Jaffe C L , Ferguson M A J , McConville M J
Published: August 2015

Glycoinositol-phospholipids (GIPLs) are the major glycolipid class and prominant surface antigens of leishmanial parasites. The GIPLs from four serologically distinct Old World strains of Leishmania were characterized to determine inter- and intra-specific differences in these glycolipids. These studies showed that: (1) the major GIPLs of Leishmania topica (LRC-L36) and Leishmania aethiopica (LRC-L495) belong to the alpha-mannose-terminating GIPL series (iM2, iM3 and iM4) that are structurally related to the glycosyl-phosphatidylinositol anchors of both the surface proteins and the abundant lipophosphoglycan (LPG). In contrast, the GIPLs from two Leishmania major strains (LRC-L456 and LRC-L580) belong to the alpha-galactose-terminating GIPL series (GIPL-1, -2 and -3) that are more structurally related to the LPG anchor; (2) the GIPL profiles of the L. major strains differed in that a significant proportion of the GIPL-2 and -3 species (approximately 40% and 80%, respectively) in LRC-L580 are substituted with a glucose-1-PO4 residue, while this type of substitution was not detected in LRC-L456; and (3) all the GIPLs contained either an alkylacyl- or a lysoalkyl-phosphatidylinositol lipid moiety. However, the alkyl chain compositions of different GIPLs within the same strain was variable. In L. major, the major GIPL species contained alkylacylglycerols with predominantly C18:0 and C24:0 alkyl chains, whereas the glucose-1-PO4-substituted GIPLs contained exclusively lysoalkylglycerols with C24:0 alkyl chains. In L. tropica, the major GIPL, iM2, contained predominantly C24:0 alkyl chains whereas the structurally related iM3 and iM4 GIPLs in this strain contained predominantly C18:0 alkyl chains. In L. aethiopica all the GIPLs (iM2, iM3, iM4) contained C18:0 alkyl chains. These data suggest that the synthesis of the GIPLs may occur in more than one subcellular compartment. The possibility that species-specific differences in the predominantly surface glycan structures may modulate the interaction of the parasite with the insect and mammalian hosts is discussed.
Expression of rat brain nitric oxide synthase in baculovirus-infected insect cells and characterization of the purified enzyme
Authors: Harteneck C , Klatt P , Schmidt K , Mayer B
Published: August 2015

Rat brain nitric oxide synthase was expressed to a high level in baculovirus-infected insect cells and purified to apparent homogeneity by affinity chromatography. The enzyme had a specific activity of approximately 1 mumol of citrulline.min-1.mg of protein-1 and contained 0.93, 0.45, 0.18 and 0.23 mol of haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin), FAD and FMN per mol of subunit respectively.
Specific activation of β-casein kinase by the inflammatory cytokines interleukin 1 and tumour necrosis factor
Authors: Guesdon F , Waller R J , Saklatvala J
Published: August 2015

Increases (5-fold) in the rate of phosphorylation of beta-casein were observed in extracts of human gingival fibroblasts that had been stimulated by interleukin 1 (IL-1) or tumour necrosis factor (TNF). The induced kinase was cytosolic and had little activity on alpha-casein. Its chromatographic behaviour on anion-exchange and gel-filtration columns was similar to that of beta-casein kinase, an enzyme detected originally in MRC-5 cells stimulated by IL-1 and TNF. Phosphopeptide maps of beta-casein confirmed that the kinase activated in gingival fibroblasts had the same substrate specificity as beta-casein kinase. In gingival fibroblasts, beta-casein kinase activity was maximum after 15 min of stimulation by IL-1 or TNF, and remained activated for several hours. Activations of small heat-shock protein (hsp27) kinase and mitogen-activated protein (MAP) kinase were also maximum 15 min after stimulation, but decreased to background levels within the next 30 min. Study of the effects of 21 agents other than IL-1 or TNF showed that none activated beta-casein kinase, whereas several activated MAP kinase or hsp27 kinase. beta-Casein kinase was also detected in extracts of bovine articular chondrocytes and human endothelial cells stimulated by IL-1 or TNF. Semi-purified preparations of fibroblast beta-casein kinase were not inactivated by phosphatases in vitro. Our results suggest that it may be involved in responses specific to IL-1 and TNF in a wide range of cell types and that its activation probably involves mechanisms other than its phosphorylation.
Incorporation of carbon from photosynthetic products into 2-carboxyarabinitol-1-phosphate and 2-carboxyarabinitol
Authors: Andralojc P J , Dawson G W , Parry M A J , Keys A J
Published: August 2015

The synthesis of 2-carboxy-D-arabinitol-1-phosphate (CA1P), the naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, was studied in leaves of the French bean Phaseolus vulgaris, L. Leaves were supplied with air containing 14CO2 in the light then the plants were transferred to normal air in the light or in the dark. Leaf samples were frozen in liquid nitrogen, ground to a powder and extracted with acid. Lipids, pigments and cations were removed from the extract and CA1P and 2-carboxy-D-arabinitol (CA) recovered by anion exchange chromatography. The CA1P was further purified by its specific binding to purified ribulose-1,5-bisphosphate carboxylase/oxygenase. CA and CA1P were identified by chromatographic properties and n.m.r. spectra. When plants were kept for 15 h in darkness after exposure to 14CO2, up to 2.2% and 5.5% of the radioactivity in the extracts was present in CA1P and CA, respectively. The most radioactivity appeared in these compounds when photosynthesis from 14CO2 took place at low photosynthetic photon flux density (PPFD). Under such conditions, radioactivity was detected in CA1P after only 10 min. During subsequent exposure to normal air (12CO2) at low PPFD the amount of radioactivity in CA1P remained almost constant for 6 h; in darkness the rate of incorporation of radioactivity into CA1P reached a maximum after 2 h and the radioactivity was still increasing 6 h later. At low PPFD, the amount of CA1P in the leaves reached a maximum after 2 h. In darkness, the amount of CA1P began to increase rapidly after a lag of almost 1 h, well ahead of the increase in radioactivity in CA1P.
The gene family of EF-hand calcium-binding proteins from the flagellum of Trypanosoma brucei
Authors: Wu Y , Deford J , Benjamin R , Lee M G , Ruben L
Published: August 2015

The flagellum of Trypanosoma brucei contains calmodulin, and a separate family of antigenically related EF-hand calcium-binding proteins which we call calflagins. The following study evaluates the structure and genomic organization of the calflagin family. Genomic Southern blots indicated that multiple copies of calflagin genes occurred in T. brucei, and that all of these copies were contained in a single 23 kb XhoI-XhoI fragment on chromosomes 15 and 16 mRNAs of 1.2 and 1.6 kb were identified in bloodstream and procyclic life-cycle stages. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. The calflagin genes were arranged tandemly along the genomic fragments. Three new members of the calflagin family were sequenced from a cDNA clone and the two genomic clones. Two unrelated families of 3′ flanking sequences were downstream from the calflagin genes. An open reading frame that was unrelated to any calflagin sequence was at the 5′ end of the 2.5 kb genomic fragment. The deduced amino acid sequences of the genomic clones (called Tb-24 and Tb-1.7g) were similar to the previously described Tb-17. Each encoded an approximately 24 kDa protein which contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. The cDNA encoded a protein (called Tb-44A) which was approximately twice as large as the other calflagins. The large size resulted from a nearly direct repeat of 186 amino acids. In general, variability among the T. brucei calflagins was greater than observed for related proteins from Trypanosoma cruzi. We demonstrate that this variability resulted from amino acid substitutions at the N-terminus, C-terminal extensions, and duplication of internal segments.
Effect of protamine on lipoprotein lipase and hepatic lipase in rats
Authors: Hultin M , Olivecrona G , Olivecrona T
Published: August 2015

The polycation protamine impedes the catabolism of triglyceride-rich lipoproteins and this has been suggested to be due to intravascular inactivation of lipoprotein lipase. We have made intravenous injections of protamine to rats and found that both lipoprotein lipase and hepatic lipase activities were released to plasma. The effect of protamine was more short-lived than that obtained by injection of heparin. The release of hepatic lipase by protamine was as effective as the release by heparin, while the amount of lipoprotein lipase released by protamine was only about one-tenth of that released by heparin. This was not due to inactivation of lipoprotein lipase, since injection of an excess of heparin 10 min after injection of protamine released as much lipoprotein lipase activity to plasma as in controls. The results in vivo differed from those obtained in model experiments in vitro. Protamine was able to almost quantitatively release both lipoprotein lipase and hepatic lipase from columns of heparin-agarose. The displacement was dependent on the total amount of protamine that had passed over the column, indicating that it was due to occupation by protamine of all available binding sites. Our results in vivo showed that the binding sites for lipoprotein lipase were not blocked as efficiently as those for hepatic lipase, indicating that the binding structures were not identical. It was concluded that the impaired turnover of lipoproteins by protamine probably was due to prevention of binding of the lipoproteins to endothelial cell surfaces rather than to impaired lipase function.
Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase
Authors: Shepherd P R , Navé B T , Siddle K
Published: August 2015

We have investigated the involvement of phosphoinositide (PI) 3-kinase and p70 ribosomal protein-S6 kinase (p70s6k) in mediating insulin stimulation of glycogen synthesis in 3T3-L1 adipocytes using specific inhibitors. Wortmannin inhibited PI 3-kinase activity (IC50 approximately 10 nM), inhibition being complete at 100 nm. Wortmannin (100 nM) completely blocked the ability of insulin to activate glycogen synthase in 3T3-L1 adipocytes and the ability of insulin to stimulate glucose incorporation into glycogen in 3T3-L1 fibroblasts. Rapamycin, which blocks insulin-stimulated activation of p70s6k, decreased insulin activation of glycogen synthase in a dose-dependent manner (IC50 approximately 0.8 ng/ml), with a maximum approx. 75% inhibition of insulin's stimulatory effect. Rapamycin inhibited insulin-stimulated glucose incorporation into glycogen to a similar extent and with similar dose-dependency, while having no effect on insulin-stimulated glucose transport. We conclude that PI 3-kinase and p70s6k are involved in the signalling pathways by which insulin stimulates glycogen synthase in 3T3-L1 adipocytes.