bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulphation of proteochondroitin and 4-methylumbelliferyl β-d-xyloside-chondroitin formed by mouse mastocytoma cells cultured in sulphate-deficient medium
Authors: Silbert J E , Sugumaran G , Cogburn J N
Published: August 2015

Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3′-phosphate 5′-phosphosulphate.
Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector
Authors: Sivaprasadarao A , Findlay J B C
Published: August 2015

In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1. However, this construct did not generate detectable expression of RBP in E. coli. When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm. Deletion of the 3′ untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx. 20-fold). After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin. The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e. it interacted with retinol, transthyretin and its cell-surface receptor. Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher. This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.
Cytotoxicity of nitric oxide in Fu5 rat hepatoma cells: evidence for co-operative action with hydrogen peroxide
Authors: Ioannidis I , de Groot H
Published: August 2015

The NO-releasing compounds 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) mediated a rapid loss of viability of Fu5 rat hepatoma cells. SIN-1 in addition to NO also released the superoxide anion radical (O2-.). Its cytotoxicity, however, was not affected by superoxide dismutase. In contrast, the H2O2-converting enzyme catalase significantly, but not completely, diminished cell damage, indicating participation of H2O2 in the tumoricidal activity of SIN-1. Glucose oxidase (5 m-units/ml), producing similar amounts of H2O2 to 5 mM SIN-1, had no effect on cell viability. When 5 m-units/ml glucose oxidase was added to incubations with 5 mM SNP, which alone initiated cell injury of about 40%, cell damage was significantly increased up to 95%. Similar results were observed with 1 mM SNAP and 20 m-units/ml xanthine oxidase, which mediated cytotoxicity of about 90% when both compounds were added together, compared with 35% and 55% cell injury, respectively, induced by the single compounds. The results indicate that a co-operative action with H2O2 enhances the tumoricidal activity of NO in Fu5 cells. No evidence for an interplay of NO with O2-. in cytotoxicity, e.g. via the peroxynitrite anion (ONOO-), was found.
Kinetics of enzymes with iso-mechanisms: analysis of product inhibition
Authors: Rebholz K L , Northrop D B
Published: August 2015

Isomerizations of free enzyme can be detected in kinetic patterns of product inhibition when the isomerization is partially rate-limiting. The kinetic pattern is non-competitive, owing to binding of substrate and product to different forms of free enzyme. This adds an additional term to the rate equation, sometimes represented as KSP. Several kineticists have noted that, as the rate of isomerization becomes high in relation to catalytic turnover, the intercept effect will become small, KSP will approach infinity, and the pattern will look competitive. Britton [(1973) Biochem. J. 133, 255-261] asserted that KSP will also approach infinity when the rate of isomerization becomes low. This second assertion is incorrect and can be traced to the particular model and graphical representation used to examine KSP as a function of relative rate constants. The function portrayed as a parabola with two roots for KSP is, instead, a straight line with one root. The algebraic condition justifying the second root obtains in the limit of zero in the rate of reaction and thus is not experimentally relevant, and the appearance of competitive inhibition, based on KSP alone, is not valid. Using a more general model, new equations are derived and presented which provide direct calculations of the apparent rate constants for free enzyme isomerizations from product-inhibition data when the equilibrium of the isomerization is near 1, and useful limits for the rate constants when greater than or less than 1.
Functional, spectroscopic and structural properties of haemoglobin from chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex)
Authors: Ascenzi P , Clementi M E , Condò S G , Coletta M , Petruzzelli R , Polizio F , Rizzi M , Giunta C , Peracino V , Giardina B
Published: August 2015

The functional and spectroscopic properties of chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex) haemoglobin (Hb) have been studied with special reference to the action of allosteric effectors and temperature. Moreover, the amino acid sequences of the N-terminal segments of the alpha- and beta-chains have been determined. The present results indicate that chamois and steinbock Hbs display a low affinity for O2, which appears to be modulated in vivo by Cl- ions rather than 2,3-bisphosphoglycerate. The Bohr effect for O2 binding to chamois and steinbock Hb is higher than for reindeer and bovine Hbs, being similar to that of human Hb. Moreover, the temperature-dependence of oxygenation appears intermediate between that of human and reindeer Hbs. E.p.r. and absorption spectroscopic properties of the ferrous nitrosylated derivative of chamois and steinbock Hbs suggest that both haemoproteins are in a low-affinity conformation even in the absence of InsP6. The reduced effect of polyphosphates on the functional and spectroscopic properties of chamois and steinbock Hb agree with amino acid differences in the N-terminal segment of the beta-chains (i.e. the deletion of Val(NA1) and the replacement of His(NA2), present in human Hb, and Gln(NA2), present in horse Hb, by Met). The molecular mechanism modulating the basic reaction of O2 with chamois and steinbock Hb may be linked to specific physiological needs related to the high-altitude habitats of these two animals.
Correlation of apoptosis with change in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II)
Authors: Zalewski P D , Forbes I J , Betts W H
Published: August 2015

Zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)-acetic acid], a membrane-permeant fluorophore specific for Zn(II), was used with spectrofluorimetry and video image analysis to reveal and quantify labile intracellular Zn. Zinquin labelled human chronic-lymphocytic-leukaemia lymphocytes, rat splenocytes and thymocytes with a weak diffuse fluorescence that was quenched when intracellular Zn was chelated with NNN‘N’-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and was greatly intensified by pretreatment of cells with the Zn ionophore pyrithione and exogenous Zn. There was substantial heterogeneity of labile Zn among ionophore-treated cells, and fluorescence was largely extranuclear. The average contents of labile Zn in human leukaemic lymphocytes, rat splenocytes and rat thymocytes were approx. 20, 31 and 14 pmol/10(6) cells respectively. Morphological changes and internucleosomal DNA fragmentation indicated substantial apoptosis in these cells when the level of intracellular labile Zn was decreased by treatment with TPEN. Conversely, increasing labile Zn by pretreatment with Zn plus pyrithione suppressed both spontaneous DNA fragmentation and that induced by the potent apoptosis-induced agents colchicine and dexamethasone. These results suggest that prevention of apoptosis is a function of labile Zn, and that a reduction below a threshold concentration in this Zn pool induces apoptosis.
Flux control coefficients determined by inhibitor titration: the design and analysis of experiments to minimize errors
Authors: Small J R
Published: August 2015

This paper is a study into the effects of experimental error on the estimated values of flux control coefficients obtained using specific inhibitors. Two possible techniques for analysing the experimental data are compared: a simple extrapolation method (the so-called graph method) and a non-linear function fitting method. For these techniques, the sources of systematic errors are identified and the effects of systematic and random errors are quantified, using both statistical analysis and numerical computation. It is shown that the graph method is very sensitive to random errors and, under all conditions studied, that the fitting method, even under conditions where the assumptions underlying the fitted function do not hold, outperformed the graph method. Possible ways of designing experiments to minimize the effects of experimental errors are analysed and discussed.
A comparison of Zn(II) and Co(II) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme
Authors: Wu H B , Tsou C L
Published: August 2015

The kinetics of reconstitution of apoacylase with either Zn(II) or Co(II) and the inactivation of the Co(II) reconstituted enzyme by 1,10-phenanthroline (OP) has been studied by following the substrate reaction continuously in presence of the metal ion or OP respectively. Although the native Zn(II)-containing and the Co(II)-reconstituted enzymes have closely similar Michaelis constants and maximal velocities, the kinetics for both the inactivation by OP and the reconstitution of the apoenzyme with the metal ions differs considerably. For Co(II), both the inactivation by OP and the reconstitution show simple kinetics, but for Zn(II), the inhibition by OP is a multi-phasic process [Wang, Wu, Wang, Zhou and Tsou (1992) Biochem. J. 281, 285-290], and the kinetics of reconstitution is also much more complicated. Both the native and the Co(II)-reconstituted enzymes are inhibited by excess of Zn(II), but not by Co(II). The inhibition by Zn(II) in excess and the reconstitution of the apoenzyme with Zn(II) are co-operative processes. The inhibition by Zn and its effect on the fluorescence emission of 1-anilinonaphthalene-8-sulphonic acid bound to the native enzyme indicate multiple Zn(II)-binding sites.
Relocalization of Drosophila dorsal protein can be induced by a rise in cytoplasmic calcium concentration and the expression of constitutively active but not wild-type Toll receptors
Authors: Kubota K , Keith F J , Gay N J
Published: August 2015

The generation of dorso-ventral polarity in Drosophila relies on the formation of a nuclear gradient of the rel/nuclear factor kappa B transcription factor dorsal in the pre-cellular syncitial embryo by a process of differential nuclear localization. It is thought that the gradient is formed by activation at ventral positions of the membrane receptor Toll that in turn causes the local dissociation of dorsal from the cytoplasmic anchor protein cactus. Although Toll is related in its cytoplasmic domain to the interleukin-1 receptor little is known about the signal transduction pathways that lead from Toll to the relocalization of dorsal. In this paper we have used immunofluorescence microscopy as a direct assay of dorsal protein nuclear localization in the Drosophila cell line Schneider 2. We find that increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of dorsal. By contrast, we find that activation of endogenous protein kinase A and expression of wild-type Toll receptors, which activate zen-chloramphenicol acetyltransferase reporter genes in this system, have only a marginal effect on the cellular distribution of the dorsal protein. Treatment of cells with activators of protein kinase C and radical oxygen intermediates, both of which activate nuclear factor kappa B, also has little effect on dorsal protein localization. We propose that different threshold levels of dorsal activation can be established by distinctly regulated signal transduction pathways.
Production of rat soluble and membrane-bound catechol O-methyltransferase forms from bifunctional mRNAs
Authors: Tenhunen J , Ulmanen I
Published: August 2015

In the rat, the catechol O-methyltransferase (COMT) gene has been found to contain two promoters, P1 and P2. This organization enables the gene to produce a soluble (S-COMT) and a membrane-associated (MB-COMT) protein by using two in-frame ATG initiation codons (S- and MB-ATG). The P1 promoter expresses a 1.6 kb transcript (S-mRNA) which codes for the S-COMT polypeptide only. Here we demonstrate that the P2 promoter controls the expression of alternatively spliced 1.9 kb transcripts (MB-mRNA) which differ by a 27-nucleotide region immediately upstream of the MB-AUG codon. The presence of the 27-base sequence alters the nucleotide at position -3 from G to C, thereby changing the translation initiation context of the MB-AUG codon. Expression experiments in COS-7 cells using full-length COMT cDNAs showed that this alteration affected the initiation of the translation of the MB-AUG and consequently changed the relative amounts of MB- and S-COMT polypeptides produced. No proteolytic cleavage of the MB-COMT form to S-COMT was detected in in vitro or in vivo pulse-chase experiments. We conclude that the bifunctional 1.9 kb mRNAs are able to produce both S-COMT and MB-COMT polypeptide by the leaky scanning mechanism of translation initiation.
Identification of a family of casein kinases in Paramecium: biochemical characterization and cellular localization
Authors: Walczak C E , Anderson R A , Nelson D L
Published: August 2015

Protein phosphorylation is believed to play a role in the regulation of ciliary motility in the protozoan Paramecium tetraurelia. Five protein kinases from Paramecium, activated by cyclic nucleotides or Ca2+, have been characterized previously. We report here the identification of a family of second-messenger-independent casein kinases in Paramecium. Casein kinase activity was enriched in the soluble fraction of cilia, but there was also significant activity tightly associated with axonemes. Three ciliary casein kinase activities (soluble CKS1 and CKS2, and axonemal CKA) were separated by chromatography and characterized. The native forms of all three were monomeric, with molecular masses of 28-45 kDa as judged by in-gel kinase assays and sizing by gel filtration. CKS2 was inhibited by heparin, but CKA was unaffected and CKS1 was stimulated. All three activities preferred acidic substrates such as casein and phosvitin, but they could be distinguished by their preference for other substrates. Antibodies against mammalian casein kinase I recognized CKS1 and CKS2 in immunoblots (43 kDa), but did not stain CKA. The antibodies to casein kinase I were used to probe other cellular fractions. A 65 kDa antigen (particulate casein kinase, CKP) was enriched in particulate fractions of whole cells. This 65 kDa protein was found in isolated cell cortices, but was not present in the infraciliary lattice. This report represents the first biochemical identification of a casein kinase I family in protozoa.
Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization
Authors: Cik M , Chazot P L , Stephenson F A
Published: August 2015

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.
Expression of β1- and β3-adrenergic-receptor messages and adenylate cyclase β-adrenergic response in bovine perirenal adipose tissue during its transformation from brown into white fat
Authors: Casteilla L , Muzzin P , Revelli J P , Ricquier D , Giacobino J P
Published: August 2015

Possible modifications of the beta-adrenergic effector system during the development of bovine perirenal brown adipose tissue (BAT) in utero and its transformation into white-like adipose tissue after birth were studied. The parameters assessed were the level of expression of beta 1-, beta 2- and beta 3-adrenergic receptor (AR) mRNAs and the response of the plasma-membrane adenylate cyclase to (-)-isoprenaline and to the beta 3-agonist BRL 37344. The beta 3-AR mRNA was found to be expressed very early in utero, i.e. before the third month of foetal life. Then it increased dramatically (9-fold) between month 6 of foetal life and birth. A high beta 3-AR mRNA level was maintained after birth up to an age of 3 months. After conversion of BAT into white-like adipose tissue, i.e. in the adult bovine, the beta 3-AR mRNA expression became small or not detectable, and the beta 1-AR mRNA, which was expressed much less than the beta 3-AR mRNA in foetal life, became predominant. A response of the adenylate cyclase to (-)-isoprenaline was observed in foetal life (3.1-fold stimulation). It decreased after birth (1.8-fold stimulation) and then remained constant until adulthood. A response to BRL 37344 was also observed in foetal life (1.8-fold stimulation). It was maintained after birth, but disappeared in the adult. A possible relationship between the beta-AR expression and the adenylate cyclase response to (-)-isoprenaline on the one hand and the uncoupling-protein expression on the other is discussed. The bovine might represent a good model to understand the transition from brown to white fat in the human.
Control analysis applied to the whole body: control by body organs over plasma concentrations and organ fluxes of substances in the blood
Authors: Brown G C
Published: August 2015

Metabolic control analysis is adapted as a method for describing and analysing the control by organs in the body over the fluxes and concentrations of substances carried in the blood. This physiological control analysis can most usefully be applied to substances with fluxes into and out of organs that are uniquely dependent only on their plasma concentrations. The organ flux of a substance is defined as the steady-state net flux of a substance into a particular organ. The organ flux control coefficients quantify the extent to which a particular organ controls the flux of a substance into the same or another particular organ. Organ concentration control coefficients quantify the extent to which an organ controls the steady-state concentration of a substance in the blood. The control coefficients are additive and obey summation, connectivity and branching theorems. Thus the control coefficients can be determined experimentally by measuring the sensitivities (elasticities) of organ fluxes to the plasma concentration of the substance. As an example of the application of these concepts, the control of ketone-body metabolism in vivo is analysed using data from the literature.
Cyclosporin A blocks 6-hydroxydopamine-induced efflux of Ca2+ from mitochondria without inactivating the mitochondrial inner-membrane pore
Authors: Reichman N , Porteous C M , Murphy M P
Published: August 2015

Oxidative stress causes Ca(2+)-loaded mitochondria to release Ca2+. The mechanism of this efflux is unclear, but it appears to be associated with the opening of a pore in the mitochondrial inner membrane. Pore opening depolarizes the mitochondria, letting solutes enter the mitochondrial matrix, causing swelling. Cyclosporin A (CsA) prevents opening of this pore. The neurotoxin 6-hydroxydopamine (6HD) autoxidizes, producing free radicals, which cause oxidative stress. In this paper it is shown that 6HD-induced efflux from Ca(2+)-loaded mitochondria was prevented by CsA. The 6HD-induced Ca2+ efflux was not accompanied by mitochondrial swelling, depolarization of the mitochondrial inner membrane or movement of radiolabelled sucrose into the mitochondrial matrix. In agreement with others [Schlegel, Schweizer and Richter (1992) Biochem. J. 285, 65-69], these findings suggest that the mitochondrial pore remained closed during pro-oxidant-induced Ca2+ efflux. However, the implication that CsA blocks pro-oxidant-induced Ca2+ efflux by some mechanism other than inactivating the mitochondrial pore, suggests that the interaction of CsA with mitochondria may be more complex than is currently supposed.
Plasmin modulates the thrombin-evoked calcium response in C6 glioma cells
Authors: Turner J S , Redpath G T , Humphries J E , Gonias S L , Vandenberg S R
Published: August 2015

Extracellular proteinases may be selectively targeted to cell surfaces by specific receptors or binding sites. In previous studies, we have characterized cellular binding sites for plasminogen and plasmin on rat C6 glioma cells. In this investigation, we studied the response of C6 cells to alpha-thrombin and plasmin by measuring the rapid kinetics of free intracellular Ca2+ concentrations ([Ca2+]i). Thrombin produced a strong, concentration-dependent rise in [Ca2+]i with an onset within 3 s and peak levels achieved in less than 10 s. A similar response was also evoked by an SFLLRN-containing thrombin-agonist peptide. C6 cells did not respond to plasmin (25 nM-1.5 microM). By contrast, pretreatment of C6 cells with 100 nM plasmin significantly inhibited the [Ca2+]i response to thrombin and the thrombin-agonist peptide. The peak [Ca2+]i response to thrombin, in cells pretreated with plasmin, was reduced by approx. 50%. The effect of plasmin on the cellular response to thrombin was selective, as pretreatment of the cells with plasmin did not affect the [Ca2+]i response to platelet-activating factor. Di-isopropylphosphorylplasmin and plasminogen did not inhibit the cellular response to thrombin, indicating that plasmin activity is required and that occupancy of cellular plasmin(ogen)-binding sites alone is insufficient. These studies demonstrate that plasmin does not directly induce a response in C6 cells, but may affect cellular function by specifically modulating the thrombin response.
In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells
Authors: Patton J R
Published: August 2015

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.
Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca2+-ATPase and Rap1B in platelets and various cell lines
Authors: Magnier C , Bredoux R , Kovacs T , Quarck R , Papp B , Corvazier E , de Gunzburg J , Enouf J
Published: August 2015

Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylation of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. As this transport was recently found to be controlled by two isoforms of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA), the 100 kDa SERCA2b and the newly identified 97 kDa SERCA, we attempted to establish which isoform is involved in this regulation. For this purpose, we studied the expression and regulation of both the SERCA and Rap1 isoforms in platelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determined by detection of its phosphoenzyme formation and by Western blotting using an isoform-specific antibody. In contrast, the expression of the 97 kDa SERCA, studied by the same methods, varied from total absence in the cancer cells to high levels in the megakaryocytic cell lines. With regard to the potential regulatory Rap1 proteins, Western blotting showed different expression of total Rap1 isoforms among the cell lineages, thus ruling out any possible relationship between Rap1 and SERCA2b. However, the expression of Rap1 proteins correlated with that of the 97 kDa SERCA isoform. More refined analysis of the rap1A and rap1B isoforms by reverse transcription PCR and by determining cAMP-induced phosphorylation of Rap1B, i.e. its functional mechanism, confirmed the correlation between Rap1B and the 97 kDa SERCA expression. This relationship was also established by the concerted up-regulation of these two proteins demonstrated in the pathological model of platelets from hypertensive rats. It is concluded that the expressions of 97 KDa SERCA and Rap1B are related, suggesting that regulation of the platelet Ca(2+)-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.
Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase
Authors: Zhou L , Xue G P , Orpin C G , Black G W , Gilbert H J , Hazlewood G P
Published: August 2015

The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.
Two sites of glucose control of insulin release with distinct dependence on the energy state in pancreatic B-cells
Authors: Detimary P , Gilon P , Nenquin M , Henquin J C
Published: August 2015

The energy state of pancreatic B-cells may influence insulin release at several steps of stimulus-secretion coupling. By closing ATP-sensitive K+ channels (K(+)-ATP channels), a rise in the ATP/ADP ratio may regulate the membrane potential, and hence Ca2+ influx. It may also modulate the effectiveness of Ca2+ on its intracellular targets. To assess the existence of these two roles and determine their relative importance for insulin release, we tested the effects of azide, a mitochondrial poison, on mouse B-cell function under various conditions. During stimulation by glucose alone, when K(+)-ATP channels are controlled by cellular metabolism, azide caused parallel, concentration-dependent (0.5-5 mM), membrane repolarization, decrease in cytosolic Ca2+ concentration [Ca2+]i and inhibition of insulin release. When K(+)-ATP channels were closed pharmacologically (by tolbutamide in high glucose), azide did not repolarize the membrane or decrease [Ca2+]i, and was much less effective in inhibiting insulin release. A similar resistance to azide was observed when K(+)-ATP channels were opened by diazoxide, and high K+ was used to depolarize the membrane and raise [Ca2+]i. In contrast, azide similarly decreased ATP levels and increased ADP levels, thereby lowering the ATP/ADP ratio under all conditions. In conclusion, lowering the ATP/ADP ratio in B-cells can inhibit insulin release even when [Ca2+]i remains high. However, this distal step is much more resistant to a decrease in the energy state of B-cells than is the control of membrane potential by K(+)-ATP channels. Generation of the signal triggering insulin release, high [Ca2+]i, through metabolic control of membrane potential requires a higher global ATP/ADP ratio than does activation of the secretory process itself.
Biosynthesis of two distinct types of mucin in HM3 human colon cancer cells
Authors: Ohara S , Byrd J C , Gum J R , Kim Y S
Published: August 2015

Mucins, high-M(r) glycoproteins with a large amount of O-glycosidically linked carbohydrate, protect the colonic epithelial surface and are altered in ulcerative colitis and colon cancer. At least two mucin genes, MUC2 and MUC3, are expressed at high levels in the human intestine. As an experimental model for studying the biosynthesis of human intestinal mucins, we used HM3 colon cancer cells. When mature mucins labelled with [3H]glucosamine or [3H]threonine were analysed by gel filtration, it was found that secreted mucins (M(r) > 10(8) were larger than soluble cellular mucins (M(r) approx. 5 x 10(6)). Only secreted mucin was sensitive to reduction. Both MUC2 and MUC3 proteins, identified by labelling with [3H]threonine or [35S]cysteine and immunoprecipitation with antibodies to synthetic mucin peptides, were already of large size (M(r) > 180,000) by the earliest labelling time (5 min). The MUC3 precursor was completely degraded by trypsin, but the MUC2 precursor had a trypsin-resistant fragment of M(r) approx. 240,000 containing threonine and cysteine. The trypsin-resistant MUC2 fragment contained N-linked carbohydrate, as indicated by a decrease in size as a result of peptidyl N-glycosidase digestion or tunicamycin treatment of HM3 cells. These results show that HM3 colon cancer cells produce at least two distinct human intestinal mucins. They also indicate that the mechanisms of biosynthesis of intestinal mucins differ from those of other mucin-like glycoproteins that have been studied.
NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization
Authors: Nisimoto Y , Otsuka-Murakami H , Iwata S
Published: August 2015

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2′,5′-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.
Binding of Ca2+ to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies
Authors: Henderson I M , Khan Y M , East J M , Lee A G
Published: August 2015

Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1′Ca2. The E1 conformation of the ATPase initially has one high-affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1′Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1′Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the ‘gating’ site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.
Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080)
Authors: Fertala A , Sieron A L , Ganguly A , Li S W , Ala-Kokko L , Anumula K R , Prockop D J
Published: August 2015

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.
Purification and properties of a phenol sulphotransferase from Euglena using l-tyrosine as substrate
Authors: Saidha T , Schiff J A
Published: August 2015

A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var. bacillaris. The apparent Km for adenosine 3′-phosphate 5′-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5′-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km values. Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine. D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position. Adenosine 3′,5′-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (Ki = 20 microM) and uncompetitive (Ki = 500 microM) inhibition kinetics respectively. This appears to be the first phenol sulphotransferase to accept tyrosine as substrate. This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.
Identification of targeting proteinase for rat α1-macroglobulin in vivo. Mast-cell tryptase is a major component of the α1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes
Authors: Tsuji A , Akamatsu T , Nagamune H , Matsuda Y
Published: August 2015

The alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes was purified by a series of column chromatographic steps on concanavalin A-Sepharose, Sephacryl S-300, DEAE-cellulose and TSK gel DEAE-5PW columns. The complex contained no detectable alpha 2-macroglobulin. Studies on the substrate specificity indicated that the complex had tryptase-like activities towards various synthetic substrates, but no elastase, chymotrypsin, cathepsin-B and cathepsin-L activities. The proteinase activity was completely inhibited by di-isopropyl fluorophosphate, leupeptin and antipain, indicating that the proteinase bound to alpha 1-macroglobulin is a serine proteinase. Two protein bands (62 and 59 kDa) of the complex were labelled with [3H]diisopropyl fluorophosphate and both bands cross-reacted with anti-(mast-cell tryptase)antibody. These results suggest that mast-cell tryptase is a major targeting proteinase for alpha 1-macroglobulin in vivo. The main alpha-macroglobulin-proteinase complex in the adjuvant-treated rats was also the alpha 1-macroglobulin-tryptase complex, even though the plasma level of alpha 2-macroglobulin was elevated.
Regulation of lipoprotein lipase activity and mRNA in the mammary gland of the lactating mouse
Authors: Jensen D R , Gavigan S , Sawicki V , Witsell D L , Eckel R H , Neville M C
Published: August 2015

We examined the effects of reproductive stage and fasting on lipoprotein lipase (LPL) activity and mRNA in the mouse mammary gland. Heparin-releasable and cell-associated LPL activity rose immediately after birth, followed 1-2 days later by an increase in LPL mRNA. Fasting decreased LPL activity in the mammary gland at all reproductive stages. During lactation, both milk and heparin-releasable LPL were substantially decreased by an overnight fast, whereas cell-associated LPL was less affected and LPL mRNA did not change. These studies indicate that the extracellular, heparin-releasable, fraction of mammary LPL activity responds most rapidly to alterations in physiological state, usually accompanied by smaller changes in cellular enzyme activity. Changes in the level of LPL mRNA were seen only during the transition from pregnancy to lactation, and these tended to follow, rather than precede, changes in enzyme activity. We conclude that in the mammary gland as in adipose tissue, LPL is regulated primarily at the translational and post-translational level.
Transcriptional regulation of the tartrate-resistant acid phosphatase (TRAP) gene by iron
Authors: Alcantara O , Reddy S V , Roodman G D , Boldt D H
Published: August 2015

Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5′ flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5′-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5′ promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881 bp of the TRAP promoter gave only a 1.5- to 2-fold increase of luciferase activity with FeTF. In all cases, increase of luciferase activity was blocked by desferrioxamine. Cells transfected with another luciferase construct driven by a simian virus 40 promoter did not show any increase of luciferase activity with FeTF. These data indicate that expression of TRAP is regulated by iron and that this regulation is exerted at the level of gene transcription. The transfection experiments also suggest that the region of the TRAP 5′-flanking sequence between base pairs -1846 and -1240 contains an iron regulatory element.