bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement
Authors: Hughes T R , Piddlesden S J , Williams J D , Harrison R A , Morgan B P
Published: August 2015

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.
Characterization of specific binding sites of 3H-labelled platelet-activating factor ([3H]PAF) and a new antagonist, [3H]SR 27417, on guinea-pig tracheal epithelial cells
Authors: Herbert J M
Published: August 2015

Binding of 3H-labelled platelet-activating factor ([3H]PAF) to guinea-pig tracheal epithelial cells was time-dependent, reversible and saturable. Scatchard analysis of the saturation-binding data indicated that [3H]PAF bound to one class of specific binding sites with high affinity (KD = 4.3 +/- 0.03 nM; Bmax. = 0.172 +/- 0.02 fmol/10(5) cells; n = 3). Unlabelled PAF competitively and selectively inhibited the specific binding of [3H]PAF with 50% inhibition at 4.8 +/- 0.07 nM (n = 3). SR 27417, the first member of a newly developed PAF antagonist series, competitively displaced [3H]PAF from its binding sites on guinea-pig tracheal epithelial cells with a Ki of 100 +/- 3 pM (n = 3). Studies carried out in parallel demonstrated that SR 27417 was 40 times more potent than C16-PAF itself and more than 100-fold as active as the best synthetic PAF-receptor antagonist yet described. [3H]SR 27417 displayed high-affinity, specific, reversible as well as saturable binding to a single class of binding sites on tracheal epithelial cells (KD = 94 +/- 7 pM; Bmax. = 0.181 +/- 0.04 fmol/10(5) cells; n = 3). C16-PAF, lyso-PAF, enantio-PAF, SR 27417 and other PAF-receptor antagonists had Ki values which were nearly identical for both [3H]PAF and [3H]SR 27417, demonstrating that in guinea-pig tracheal epithelial cells they have the same binding sites. In conclusion, these data suggest that tracheal epithelial cells contain PAF-specific receptors and indicate that SR 27417 is an extremely potent PAF-receptor antagonist, as well as being a suitable radioligand for labelling PAF receptors on intact cells.
Transport of l-glutamine and l-glutamate across sinusoidal membranes of rat liver. Effects of starvation, diabetes and corticosteroid treatment
Authors: Low S Y , Taylor P M , Hundal H S , Pogson C I , Rennie M J
Published: August 2015

There is increasing evidence that membrane transporters for glutamine and glutamate are involved in control of liver metabolism in health and disease. We therefore investigated the effects of three catabolic states [starvation (60 h), diabetes (4 days after streptozotocin treatment) and corticosteroid (8-day dexamethasone) treatment] associated with altered hepatic amino acid metabolism on the activity of glutamine and glutamate transporters in sinusoidal membrane vesicles from livers of treated rats. In control preparations, L-[14C]glutamine uptake was largely Na(+)-dependent, but L-[14C]glutamate uptake was largely Na(+)-independent. Vmax. values for Na(+)-dependent uptake of glutamine and/or glutamate exceeded control values (by about 2- and 12-fold respectively) in liver membrane vesicles from starved (glutamine), diabetic (glutamate) or steroid-treated (glutamine and glutamate) rats. The Km values for Na(+)-dependent transport of glutamine or glutamate and the rates of their Na(+)-independent uptake were not significantly altered by any treatment. Na(+)-independent glutamate uptake appeared to include a dicarboxylate-exchange component. The patterns of inhibition of glutamine and glutamate uptake by other amino acids indicated that the apparent induction of Na(+)-dependent amino acid transport in catabolic states included increased functional expression of systems A, N (both for glutamine) and X-ag (for glutamate). The results demonstrate that conditions resulting in increased secretion of catabolic hormones (e.g. corticosteroid, glucagon) are associated with increased capacity for Na(+)-dependent transport of amino acids into liver cells from the blood. The modulation of hepatic permeability to glutamine and glutamate in these situations may control the availability of amino acids for intrahepatic metabolic processes such as ureagenesis, ammonia detoxification and gluconeogenesis.
Pyridoxal arsenate as a prosthetic group for aspartate aminotransferase
Authors: Ali Bassam R. S. , Dixon Henry B. F.
Published: August 2015

The apoenzyme of aspartate aminotransferase formed a stable, active holoenzyme on treatment with pyridoxal in the presence of arsenate.
The lipolysis/esterification cycle of hepatic triacylglycerol. Its role in the secretion of very-low-density lipoprotein and its response to hormones and sulphonylureas
Authors: Wiggins D , Gibbons G F
Published: August 2015

In hepatocyte cultures maintained in the absence of extracellular fatty acids, at least 70% of the secreted very-low-density lipoprotein (VLDL) triacylglycerol was derived via lipolysis of intracellular triacylglycerol. This proportion was unchanged when the cells were exposed for 24 h to insulin or glucagon, hormones which decreased the overall secretion of intracellular triacylglycerol, or to chloroquine or tolbutamide, agents which inhibit lysosomal lipolysis. The rate of intracellular lipolysis was 2-3-fold greater than that required to maintain the observed rate of triacylglycerol secretion. Most of the fatty acids released were returned to the intracellular pool. Neither insulin nor glucagon had any significant effect on the overall lipolysis and re-esterification of intracellular triacylglycerol. In these cases a greater proportion of the released fatty acids re-entered the cellular pool, rather than being recruited for VLDL assembly. Tolbutamide inhibited intracellular lipolysis, but suppressed VLDL secretion to a greater extent. 3,5-Dimethylpyrazole did not affect lipolysis or VLDL secretion. The increased secretion of VLDL triacylglycerol observed after exposure of cells to insulin for 3 days was not accompanied by an increased rate of intracellular lipolysis. However, a larger proportion of the triacylglycerol secreted under these conditions may not have undergone prior lipolysis.
Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ
Authors: Vercesi A E , Docampo R
Published: August 2015

The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of Leishmania donovani promastigotes in situ using safranine O. L. donovani mitochondria were able to build up and retain a membrane potential of a value comparable with that of mammalian mitochondria. The response of promastigotes mitochondrial membrane potential to phosphate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), valinomycin and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria with regard to the properties of their electrochemical proton gradient. When L. donovani promastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM). The presence of 1 microM-FCCP decreased by about 75% the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably by the endoplasmic reticulum, was completely inhibited by 500 microM-vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased by about 25% and the Ca2+ set point was increased to 0.7 microM. The succinate-dependence and FCCP-and Ruthenium Red-sensitivity of the Ca2+ uptake detected in the presence of vanadate indicate that this uptake is probably by the mitochondria. This interpretation was further supported by the Ruthenium Red-sensitive decrease in the mitochondrial membrane potential caused by Ca2+ addition. The anti-leishmanial cationic drugs pentamidine and WR-6026 also induced a rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes.
Synergistic activation of phospholipase D by protein kinase C- and G-protein-mediated pathways in streptolysin O-permeabilized HL60 cells
Authors: Geny B , Cockcroft S
Published: August 2015

Stimulation of phospholipase D (PLD) by cell surface receptors has been observed in many cell types. We have investigated the mechanism of activation of this enzyme in undifferentiated HL60 cells. GTP analogues and Ca2+ (buffered in the nanomolar to micromolar range) were introduced into HL60 cells in the presence of the permeabilizing agent, streptolysin O. We report that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) is a potent activator of phospholipase D when Ca2+ is available at micromolar levels. Phorbol 12-myristate 13-acetate or Ca2+ alone can also stimulate PLD, but to a limited extent. The activation of PLD by GTP[S] can be partially dissociated from GTP[S]-stimulated phosphoinositide-specific phospholipase C, suggesting that a G-protein may be directly involved in regulating PLD. However, maximal activation of PLD only occurs under conditions that are permissive to phospholipase C stimulation. We conclude that PLD activation is under dual control, i.e. protein kinase C- as well as G-protein-mediated regulation. Synergistic activation occurs when both pathways are simultaneously stimulated. We conclude that full activation of PLD requires protein kinase C, increased Ca2+ and a GTP-binding protein. Evidence for cytosolic components that may also be involved in obtaining full activation of PLD is also presented.
Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship
Authors: Takai A , Murata M , Torigoe K , Isobe M , Mieskes G , Yasumoto T
Published: August 2015

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.
N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage
Authors: Loulakis P , Shrikhande A , Davis G , Maniglia C A
Published: August 2015

Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1′ position.
Selective inhibition of Zn2+-glycerophosphocholine cholinephosphodiesterase by tellurium tetrachloride
Authors: Sok D E , Kim M R
Published: August 2015

A Zn(2+)-glycerophosphocholine cholinephosphodiesterase (EC 3.1.4.38) purified from mouse brain was found to be reversibly inhibited by tellurium tetrachloride. This effect was characterized by a competitive pattern of inhibition, with apparent Ki values of 0.7 microM and 1.5 microM for the hydrolysis of p-nitrophenylphosphocholine and glycerophosphocholine respectively. Interestingly, the inhibitory effect of tellurium tetrachloride was found to be greatly potentiated by tetramethylammonium salt, indicative of a synergistic interaction between the two compounds. Additionally, it was observed that the effect of tellurium tetrachloride was not affected by a number of other metal ions, and was more pronounced at neutral pH, suggesting that the inhibitory role of the tellurium tetrachloride may be of importance under physiological conditions. Thus Zn(2+)-glycerophosphocholine cholinephosphodiesterase is proposed to be one of the target enzymes which is susceptible to the inhibitory effect of tellurium tetrachloride.
Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes
Authors: Anderson N G
Published: August 2015

Physiological concentrations of growth hormone induced a rapid and transient activation of mitogen-activated protein kinase (MAP kinase) and S6 kinase in 3T3-F442A preadipocytes. These effects were abrogated by staurosporine and in cells chronically pretreated with phorbol esters, suggesting that protein kinase C is involved in the mechanism of activation. In addition, three cytosolic proteins exhibited a growth-hormone-dependent increase in tyrosine phosphorylation.
Polyclonal antibody-catalysed amide hydrolysis
Authors: Gallacher G , Searcey M , Jackson C S , Brocklehurst K
Published: August 2015

1. The activated amide (4-nitroanilide), N-(4-nitrophenyl) N'-butyl-1,4-phenylenediacetamide (III) was synthesized. 2. A polyclonal antibody preparation (PCA 270-29) was elicited in a multigeneration cross-bred sheep (no. 270) and isolated 29 weeks into the immunization schedule by procedures described previously for PCA 270-22 [Gallacher, Jackson, Searcey, Badman, Goel, Topham, Mellor & Brocklehurst (1991) Biochem J. 271, 871-881]. These involved the use of an amide conjugate bonded through the carboxy group of 4-nitrophenyl 4′-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin as the immunogen. 3. PCA 270-29 was shown to catalyse the hydrolysis of both the carbonate ester substrate 4-nitrophenyl 4′-(3-aza-2-oxoheptyl)phenyl carbonate (I) and the amide substrate (III). Both catalyses obeyed the Michaelis-Menten equation with the following values of the parameters at 25 degrees C: for the hydrolysis of (I) at pH 8.0, Km = 3.96 +/- 0.28 microM and k(cat.) = 0.135 +/- 0.004 s-1 (k(non-cat.) = 1.99 x 10(-4) s-1); for the hydrolysis of (III) at pH 9.0, Km = 5.4 +/- 1.4 microM and k(cat.) = (5.95 +/- 0.75) x 10(-5) s-1 (k(non-cat.) = approx. 2 x 10(-7) s-1). 4. The finding that PCA 270-29 is almost equally effective as a catalyst for the hydrolysis of the amide (III) as for that of the carbonate ester (I) when allowance is made for the different intrinsic reactivities of the two types of substrate is discussed. The catalytic characteristics of PCA 270-29, the first example of a polyclonal catalytic antibody preparation shown to catalyse the hydrolysis of an amide and the first example of an antibody preparation (monoclonal or polyclonal) with such catalytic character to be produced by use of a phosphate immunogen, are compared with those of the small number of other antibody-mediated hydrolyses of amides in the literature.
Anaerobic reactions of Rhus vernicifera laccase and its type-2 copper-depleted derivatives with hexacyanoferrate(II)
Authors: Sakurai T
Published: August 2015

Anaerobic reactions of Rhus vernicifera laccase and its type-2 copper-depleted derivatives with hexacyanoferrate(II) were investigated by absorption and e.s.r. spectroscopy. When native laccase was treated with excess hexacyanoferrate(II), the type-1 and type-2 coppers were immediately reduced and the e.s.r. signal due to type-3 copper was transiently observed. After incubation, a novel e.s.r. signal (g parallel = 2.31, g perpendicular = 2.08) developed together with the type-1 copper signal. Only the novel e.s.r. signal was left after the sample had been treated with ascorbate. In the corresponding absorption spectrum, a new band was observed at around 490 nm. A similar new e.s.r. signal did not appear for the type-2-copper-depleted (T2D) laccase, in which the type-3 copper had been reduced during the procedure to deplete the type-2 copper. On the other hand, the novel e.s.r. signal emerged when the type-3 copper in T2D laccase had been previously reoxidized with H2O2. The novel e.s.r. signal was not significantly saturated even by 200 mV microwave power at 4 K. Quantitative estimations and a small molecule study for CuII-FeII(CN)6 and CuII-FeIII(CN)6 systems suggested that the novel e.s.r. signal corresponds to some sort of adduct involving the type-3 copper and hexacyanoferrate(II).
Variations in hepatic carbon flux during unrestricted feeding
Authors: Sugden M C , Howard R M , Holness M J
Published: August 2015

Previous findings have established a pivotal role for hepatic pyruvate dehydrogenase complex (PDH) in regulating hepatic carbon flux during the starved-to-fed and fed-to-starved nutritional transitions [Holness, McLennan, Palmer & Sugden (1988) Biochem. J. 252, 325-330; Holness & Sugden (1990) Biochem. J. 268, 77-81]. We have therefore examined liver PDH activities during the light and dark phases of the feeding cycle in the adult rat in relation to hepatic glycogenesis, fatty acid synthesis and cholesterogenesis. There was significant synchronous suppression of lipogenesis and glycogenesis during the light phase; rates were restored asynchronously during the dark (feeding) phase. Glycogen concentrations declined during the light phase and increased during the dark phase. Despite quite dramatic changes in rates of glycogen and lipid synthesis and hepatic glycogen concentrations during the light and dark phases, hepatic PDHa (active form) activity remained relatively unchanged. Qualitative and quantitative differences in the pattern of change in rates of synthesis of fatty acid and cholesterol suggested regulation at pathway-specific sites distal to PDH.
A novel mechanism for isoprenaline-stimulated proliferation of rat parotid acinar cells involving the epidermal growth factor receptor and cell surface galactosyltransferase
Authors: Purushotham K R , Dunn W A , Schneyer C A , Humphreys-Beher M G
Published: August 2015

Chronic injections of epidermal growth factor (EGF) or the beta-adrenergic receptor agonist isoprenaline resulted in rat parotid gland hypertrophy and hyperplasia. Introduction of a polyclonal antibody to EGF or the EGF-receptor (EGF-R) caused a specific retardation of acinar cell proliferation when injected along with the growth factor. Meanwhile, only the antibody to EGF-R caused a dose-dependent retardation of proliferation on co-administration with isoprenaline both in vivo and in vitro. The antibody injected alone had no effect on cell growth. When cells were incubated in the presence of EGF, plasma membranes from isoprenaline-treated and control animals showed phosphorylation of the EGF-R tyrosine moieties and transient increases in membrane-associated phospholipase C gamma. Isoprenaline did not stimulate phosphorylation of the EGF-R in isolated plasma membranes. However, activation of the phosphotyrosine-signalling pathway could be duplicated by incubating isoprenaline-treated acinar cells, but not control cells, with bovine galactosyltransferase. Immunopurified EGF-R demonstrated variations in reactivity with two different lectins after treatment of the cells with the beta-agonist as well as increased galactosyltransferase substrate capacity in vitro. In addition, incubation of intact acinar cells and isolated plasma-membrane fractions from isoprenaline-treated rats with UDP-[14C]galactose resulted in an increased incorporation of label into the EGF-R. The results suggest that the carbohydrate moiety of the EGF-R has been altered in isoprenaline-treated animals allowing galactosyltransferase now to recognize this receptor. This interaction may in part mediate proliferation of parotid acinar cells. Indeed, we have previously shown that an antibody to galactosyltransferase is capable of blocking isoprenaline-mediated acinar cell proliferation in vivo [Humphreys-Beher, Schneyer, Kidd & Marchase (1987) J. Biol. Chem. 262, 11706-11713].
Mechanism linking glycogen concentration and glycogenolytic rate in perfused contracting rat skeletal muscle
Authors: Hespel P , Richter E A
Published: August 2015

The influence of differences in glycogen concentration on glycogen breakdown and on phosphorylase activity was investigated in perfused contracting rat skeletal muscle. The rats were preconditioned by a combination of swimming exercise and diet (carbohydrate-free or carbohydrate-rich) in order to obtain four sub-groups of rats with varying resting muscle glycogen concentrations (range 10-60 mumol/g wet wt.). Pre-contraction muscle glycogen concentration was closely positively correlated with glycogen breakdown over 15 min of intermittent short tetanic contractions (r = 0.75; P less than 0.001; n = 56) at the same tension development and oxygen uptake. Additional studies in supercompensated and glycogen-depleted hindquarters during electrical stimulation for 20 s or 2 min revealed that the difference in glycogenolytic rate was found at the beginning rather than at the end of the contraction period. Phosphorylase alpha activity was approximately twice as high (P less than 0.001) in supercompensated muscles as in glycogen-depleted muscles after 20 s as well as after 2 min of contractions. It is concluded that glycogen concentration is an important determinant of phosphorylase activity in contracting skeletal muscle, and probably via this mechanism a regulator of glycogenolytic rate during muscle contraction.
Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes
Authors: Segal A W , West I , Wientjes F , Nugent J H A , Chavan A J , Haley B , Garcia R C , Rosen H , Scrace G
Published: August 2015

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.
Correction of human mucopolysaccharidosis type-VI fibroblasts with recombinant N-acetylgalactosamine-4-sulphatase
Authors: Anson D S , Taylor J A , Bielicki J , Harper G S , Peters C , Gibson G J , Hopwood J J
Published: August 2015

A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.
Effects of calmodulin antagonists on antibody binding to calmodulin. Distinct conformers of calmodulin induced by the binding of drugs
Authors: Orosz F , Liliom K , Barkhudaryan N A , Horváth L , Ovádi J
Published: August 2015

An indirect enzyme-linked immunosorbent assay has been used to study the interactions between calmodulin and two calmodulin antagonists, trifluoperazine and a neuropeptide isolated from the hypothalamus. The binding of a monospecific anti-calmodulin antibody, raised in rabbit against dinitrophenylated calmodulin, to calmodulin was tested at various concentrations of these drugs under equilibrium conditions. Trifluoperazine at low concentrations stimulated, but at relatively high concentrations inhibited, immunocomplex formation. The neuropeptide displaced the antibody from calmodulin at nanomolar concentrations. Enzyme-linked immunosorbent assays were also carried out with the large tryptic fragments of calmodulin. The results suggest that (i) the C-terminal fragment binds the antibody with an affinity which is comparable with that of intact calmodulin; (ii) the neuropeptide can form complexes with both N- and C-terminal fragments, but with two orders of magnitude less activity in case of the C-terminal fragment; and (iii) trifluorperazine does not stimulate antibody binding to the C-terminal fragment. Therefore the tertiary structure of calmodulin must be intact to ensure long-distance interactions between the binding sites of trifluoperazine, the neuropeptide and the antibody. These interactions may produce distinct conformers of calmodulin which may exhibit altered potency, not only for antibody binding but also for stimulation/inhibition of target enzymes.
Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein
Authors: van den Hombergh J P T W , Moore J D , Charles I G , Hawkins A R
Published: August 2015

The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.
Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors
Authors: Bachelot C , Cano E , Grelac F , Saleun S , Druker B J , Levy-Toledano S , Fischer S , Rendu F
Published: August 2015

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.
Improved results on generalised robust H∞ filtering for Lipschitz descriptor non‐linear systems with uncertainties
Authors: Zhang Wei , Xie Hongyang , Su Housheng , Zhu Fanglai
Published: August 2015

Recently, a generalised framework for robust non‐linear H∞ filtering of uncertain Lipschitz descriptor systems has been developed by Abbaszadeh and Marquez. The main feature of the associated H∞ filter is that it holds a general structure, which can capture both dynamic and static‐gain filter structures. In this study, the authors aim to propose improved results on the general H∞ filter synthesis for Lipschitz descriptor non‐linear systems in the presence of disturbance and model uncertainties. Based on a technique to directly deal with the Lipschitz non‐linear terms, new H∞ filter synthesis conditions that guarantee asymptotic stability of the estimation error dynamics with prescribed disturbance attention level are established and formulated in terms of linear matrix inequalities. They also give some remarks on the results of Abbaszadeh and Marquez and compare them with the author's results. More precisely, it is shown that the proposed conditions are less conservative than those in the existing results. Simulation results on an example are given to numerically verify the proposed design.
Vehicle density estimation of freeway traffic with unknown boundary demand–supply: an interacting multiple model approach
Authors: Zhang Liguo , Mao Xuerong
Published: August 2015

As distributed parameter systems, dynamics of freeway traffic are dominated by the current traffic parameter and boundary fluxes from upstream/downstream sections or on/off ramps. The difference between traffic demand–supply and boundary fluxes actually reflects the congestion level of freeway travel. This study investigates simultaneous traffic density and boundary flux estimation with data extracted from on‐road detectors. The existing studies for traffic estimation mainly focus on the traffic parameters (density, velocity etc.) of mainline traffic and ignore flux fluctuations at boundary sections of the freeway. The authors propose a stochastic hybrid traffic flow model by extending the cell transmission model with Markovian multi‐mode switching. A novel interacting multiple model filtering for simultaneous input and state estimation is developed for discrete‐time Markovian switching systems with unknown input. A freeway segment of Interstate 80 East (I‐80E) in Berkeley, Northern California, is chosen to investigate the performance of the developed approach. Traffic data is obtained from the performance measurement system.
Adaptive practical synchronisation of Lagrangian networks with a directed graph via pinning control
Authors: Ma Mihua , Cai Jianping , Zhou Jin
Published: August 2015

This study investigates the practical synchronisation of networked Lagrangian systems with a directed graph by pinning adaptive control. Some simple yet general algebraic criteria are proposed based on practical stability theory on dynamical systems, and the proposed criteria ensure that all the robotic agents described by Lagrangian dynamics can achieve desired practical synchronisation. The main features of the present investigation include, an efficient pinning adaptive strategy is implemented by applying local linear feedback injections to a small fraction of agents, which means that the presented control strategy does not require the prerequisite knowledge of system models. Moreover, an allowable attraction region is explicitly given by Lagrangian dynamics parameter and the controlled network structure, and so it is very convenient to application in practice. Besides, this study addresses the fundamental problems in the pinning control of networked Lagrangian systems: what kind of agents and how many agents should be pinned? As a direct application of the theoretical results, practical synchronisation of eight two‐link revolute manipulators is discussed in detail. Numerical experiments verify the effectiveness of the proposed control technique.
Receding horizon particle swarm optimisation‐based formation control with collision avoidance for non‐holonomic mobile robots
Authors: Lee Seung‐Mok , Myung Hyun
Published: August 2015

This study proposes a novel model predictive control (MPC) based on receding horizon particle swarm optimisation (RHPSO) for formation control of non‐holonomic mobile robots by incorporating collision avoidance and control input minimisation and guaranteeing asymptotic stability. In most conventional MPC approaches, the collision avoidance constraint is imposed by the 2‐norm of a relative position vector at each discrete time step. Thus, multi‐robot formation control problem can be formulated as a constrained non‐linear optimisation problem. In general, traditional optimisation techniques suitable for addressing constrained non‐linear optimisation problems take a longer computation time with an increase in the number of constraints. The traditional approaches therefore suffer from the computational complexity problem corresponding to an increase in the prediction horizon. To address this problem without a significant increase in computational complexity, a novel strategy for collision avoidance is proposed to incorporating a particle swarm optimisation. In addition, the stability conditions are derived in simplified forms that can be satisfied by selecting appropriate constant values for control gains and weight parameters. Numerical simulations verify the effectiveness of the proposed RHPSO‐based formation control.
Adaptive integral sliding mode control with bounded ℒ2 gain performance of uncertain quantised control systems
Authors: Zheng Bo‐Chao , Park Ju H.
Published: August 2015

Adaptive integral sliding mode control (ISMC) is investigated for a class of non‐linear uncertain systems considering the mismatch relation between quantisation sensitivity parameters. First, by analysing the operation principle of the encoder/decoder, a mismatched relation between the quantisation parameters is established by using a time‐varying ratio model, where the boundaries of the time‐varying ratio can be unknown, regarding the existing cases as special ones. Second, using linear matrix inequality techniques, the prescribed bounded ℒ2 gain performance condition is obtained for the sliding dynamics. Sequently, based on the formed boundary unknown time‐varying ratio relation, adaptive ISMC laws are designed to ensure the reachability of the sliding manifold, then the establishment of the stability of the closed‐loop system. Finally, an example is provided to illustrate the effectiveness of the proposed methods.
Properties of the generalised power theory: universality and partitioning/augmentation properties
Authors: Jeon Seong‐Jeub
Published: August 2015

The power theory, which can be applied to a power system with transmission/distribution lines with unequal resistances, has useful properties of universality and partitioning/augmentation. Its definitions for apparent power and power factor are consistent from a sinusoidal single‐phase system to a non‐sinusoidal multi‐line system. This study shows that a power system can be partitioned into sub‐power systems if some conditions are satisfied and further, it is shown that the sub‐power systems can be combined into a large power system. The application area of the concept of apparent power and power factor can be extended with consistency from a single system to a large combined system. The usefulness of the properties in analysing and designing combined power systems is illustrated using examples.
Self‐adapted single‐ended travelling wave fault location algorithm considering transfer characteristics of the secondary circuit
Authors: Xu Fei , Dong Xinzhou , Wang Bin , Shi Shenxing
Published: August 2015

The transfer characteristics of a secondary circuit consisting of a current transformer) and a secondary cable are critical to the performance of single‐ended travelling‐wave‐based fault locators. The non‐ideal nature of the secondary cable often leads to distortion of the recorded travelling waves. In addition, this may lead to wave front identification failure. A high‐frequency model of the secondary circuit is developed in this study. Based on the transfer function of the equivalent circuit, the distortion is found to be a superposition of two damped oscillation components. Recorded waveforms from an on‐site device have been used to verify the model and analysis. A self‐adapted single‐ended fault location scheme is proposed to mitigate the influence of the damped oscillation components. The dyadic wavelet transform, pre‐filtering Prony analysis and impedance fault location algorithm are used. The proposed method is tested on the recorded data, and precise fault location results are achieved. Different fault distances, fault impedances, secondary cable lengths and fault inception angles are also simulated to verify the proposed approach.
One‐day‐ahead hourly forecasting for photovoltaic power generation using an intelligent method with weather‐based forecasting models
Authors: Huang Chao‐Ming , Chen Shin‐Ju , Yang Sung‐Pei , Kuo Chung‐Jen
Published: August 2015

An intelligent method is proposed in this study to predict one‐day‐ahead hourly photovoltaic (PV) power generation. The proposed method comprises data classification, training, forecasting and forecasting updating stages. In the first stage, a fuzzy k‐means algorithm is used to classify the historical data of daily PV power generation into various weather types. In the second stage, five training models are established, according to the verbal weather forecast of the Taiwan Central Weather Bureau (TCWB), in terms such as the sunny, sunny and cloudy, cloudy, cloudy and rainy and rainy. Each training model is constructed using a radial basis function neural network (RBFNN), for which the parameters of each RBFNN, including the position of the radial basis function (RBF) centres, the width of the RBFs and the weights between the hidden and the output layers, are optimised using a harmony search algorithm (HSA). In the forecasting stage, fuzzy inference is used to select an adequate forecasting model from the trained models. To cope with the possible fluctuation of PV power generation, the forecasts are updated every 3 h, according to the updated weather forecasts of the TCWB. The proposed approach is tested on a practical PV power generation system. The results show that the proposed method provides better forecasting results than the existing methods over 1‐year testing data.
Ground distance relay compensation in the presence of delta‐hexagonal phase shifting transformer
Authors: Ghorbani Amir , Arablu Masoud
Published: August 2015

In this study, the effects of the delta‐hexagonal phase shifting transformers (PSTs) on the operation of distance relays and their trip boundaries are investigated under different fault resistances and locations. Existence of fault resistance in phase‐to‐ground faults creates an unreliable operation of distance relays. Trip boundaries are used to determine the fault resistance effects on the impedance calculated by the relay. Moreover, PSTs are used to control the power flow in transmission lines. Finally, a feasible method for eliminating the PSTs detrimental effects on the operation of the distance relays is presented. This method, which uses the voltage difference between the PST terminals, is getting prevalent in modifying the operation of the distance relays even under the high resistance faults. Phasor measurement units (PMUs) are used to calculate this voltage as PMU signals and a remote signal system is used to transmit them to the system protection centre. All the simulations are conducted with Electro‐Magnetic Transient Program.
High‐isolation microstrip patch array antenna for single channel full duplex communications
Authors: Batgerel Ariunzaya , Eom Soon Young
Published: August 2015

An orthogonally polarised stacked square microstrip patch array antenna structure with high‐isolation performance is proposed for single channel full duplex wireless communications. A double symmetric microstrip array structure is collaborated with a special self‐interference cancelling network called differential feeding network (DFN) in order to achieve high‐isolation performance. The proposed 2 × 2 and 4 × 4 symmetric array antenna structures with the DFNs were designed to operate at the idustrial, scientific and medical band of 2.34–2.54 GHz. By measurement, the average isolation performances of 53 and 60.7 dB were obtained in 200 MHz frequency band by the 2 × 2 and 4 × 4 array antennas, respectively. The measured average gains of the 2 × 2 and 4 × 4 array antennas were found to be 14.4 and 19.4 dBi, respectively.
Synthesis of a miniaturised wireless local area network dual‐band microstrip Wilkinson power divider through a general three‐step optimisation process
Authors: Gong Jian‐Qiang , Gao Kai , Liang Chang‐Hong
Published: August 2015

Synthesis method for a novel dual‐band Wilkinson power divider (DBWPD) is presented in this study. The proposed DBWPD is constructed using a novel dual‐band impedance transformer (DBIT). The DBIT is made up of a kernel circuit unit cell with uniform transmission lines attached to its two ends. A non‐linear system of equations ruling the DBIT performance is first built up and then successfully solved with numerical method. Design charts are provided for determining the feasible DBIT theoretical parameters. With the DBIT theoretical solutions, a microstrip DBWPD operative at 2.4/5.2 GHz is practically implemented, of which layout parameters are effectively resolved in a sequential three‐step optimisation design process, and its compact size as well as good dual‐band operation performance has been demonstrated by fabrication and experimental tests, confirming the proposed synthesis method.