bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory effect of fluoride on insulin receptor autophosphorylation and tyrosine kinase activity
Authors: Viñals F , Testar X , Palacín M , Zorzano A
Published: August 2015

Fluoride is a nucleophilic reagent which has been reported to inhibit a variety of different enzymes such as esterases, asymmetrical hydrolases and phosphatases. In this report, we demonstrate that fluoride inhibits tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle and human placenta. Fluoride inhibited in a similar dose-dependent manner both beta-subunit autophosphorylation and tyrosine kinase activity for exogenous substrates. This inhibitory effect of fluoride was not due to the formation of complexes with aluminum and took place in the absence of modifications of insulin-binding properties of the insulin receptor. Fluoride did not complete with the binding site for ATP or Mn2+. Fluoride also inhibited the autophosphorylation and tyrosine kinase activity of receptors for insulin-like growth factor I from human placenta. Addition of fluoride to the pre-phosphorylated insulin receptor produced a slow (time range of minutes) inhibition of receptor kinase activity. Furthermore, fluoride inhibited tyrosine kinase activity in the absence of changes in the phosphorylation of prephosphorylated insulin receptors, and the sensitivity to fluoride was similar to the sensitivity of the unphosphorylated insulin receptor. The effect of fluoride-on tyrosine kinase activity was markedly decreased when insulin receptors were preincubated with the copolymer of glutamate/tyrosine. Prior exposure of receptors to free tyrosine or phosphotyrosine also prevented the inhibitory effect of fluoride. However, the protective effect of tyrosine or phosphotyrosine was maximal at low concentrations, suggesting the interaction of these compounds with the receptor itself rather than with fluoride. These data suggest: (i) that fluoride interacts directly and slowly with the insulin receptor, which causes inhibition of its phosphotransferase activity; (ii) that the binding site of fluoride is not structurally modified by receptor phosphorylation; and (iii) based on the fact that fluoride inhibits phosphotransferase activity in the absence of alterations in the binding of ATP, Mn2+ or insulin, we speculate that fluoride binding might affect the transfer of phosphate from ATP to the tyrosine residues of the beta-subunit of the insulin receptor and to the tyrosine residues of exogenous substrates.
Intracellular maturation of the γ-carboxyglutamic acid (Gla) region in prothrombin coincides with release of the propeptide
Authors: Wallin R , Stanton C , Hutson S M
Published: August 2015

Vitamin K-dependent coagulation factors undergo several post-translational modifications before the proteins are secreted into the blood as functional zymogens of the coagulation system. The modifications include Asn-linked glycosylation, Asn/Asp hydroxylation, removal of a signal peptide for translocation of the polypeptide into the endoplasmic reticulum and removal of a propeptide which, when attached to the intracellular coagulation factor precursor, directs the protein for vitamin K-dependent gamma-carboxylation. gamma-Carboxylation of targeted Glu residues results in formation of Ca(2+)-binding gamma-carboxyglutamic acid (Gla) residues. Ca2+ binding by these residues induces a conformational change in the protein which is a necessary event for optimal activation or activity of the clotting factor in blood. In the present study we have monitored the intracellular prothrombin precursor in the secretory pathway of liver cells to determine the effect that the propeptide has on Ca(2+)-dependent folding of the protein. The data provide evidence that the Ca(2+)-induced conformational change required for activation of prothrombin coincides with release of the propeptide in the trans-Golgi apparatus of the liver cell and elucidates an important function for the endoproteinase furin in biosynthesis of vitamin K-dependent clotting factors.
Insulin-like growth factor-I and more potent variants restore growth of diabetic rats without inducing all characteristic insulin effects
Authors: Tomas F M , Knowles S E , Owens P C , Chandler C S , Francis G L , Ballard F J
Published: August 2015

The effects of graded doses of insulin-like growth factor-I (IGF-I) and two variants which bind poorly to IGF-binding proteins were investigated in 160 g streptozotocin-induced diabetic rats. The two variants were the truncated form, des(1-3)IGF-I, and another with arginine at residue 3 and an N-terminal extension, termed LR3-IGF-I. The peptides were infused via mini-osmotic pumps. Reference groups received either vehicle or insulin (30 i.u. per day). Treatment led to a marked dose-dependent increase in growth rate and nitrogen balance. The highest dose (695 micrograms/day) of IGF-I increased body weight by 48.1 +/- 1.7 g/7 days, compared with 11.0 +/- 2.8 g/7 days for the vehicle-treated group. The two variants were 2.5-3 times more potent than IGF-I in restoring growth. The insulin-treated group gained more weight (64.5 +/- 1.6 g/7 days), but the added gain was fat (92.5 +/- 4.8 g of fat/kg carcass wet wt., compared with 32.2 +/- 2.1 for all other groups) rather than protein. All peptides increased muscle protein-synthesis rates and RNA levels by up to 50%, with IGF-I the least potent. These high doses of IGFs did not decrease either the glucosuria or the daily excretion rate of N tau-methyl-histidine (N tau-MH). On the other hand, insulin treatment markedly decreased both glucosuria (from 82.7 +/- 5.4 to 4.5 +/- 3.3 mmol/day) and N tau-MH excretion (from 9.3 +/- 0.3 to 7.1 +/- 0.4 mumol/day per kg). This experiment shows that, although IGF-I and variants can restore growth in diabetic rats, other insulin-dependent metabolic processes in liver, muscle and adipose tissue are not restored.
Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells
Authors: Leamon C P , Low P S
Published: August 2015

Folate-protein conjugates have been shown to bind to and enter HeLa and KB cells by receptor-mediated endocytosis [Leamon and Low (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5572-5576]. Although these cells contain a membrane folate-binding protein (FBP) involved in the uptake of free folate, no studies have been conducted to evaluate whether the folate-protein conjugates enter cells via the same protein. To address this issue, HeLa cell monolayers were treated with folate-labelled 125I-RNAase under various conditions characteristic of FBP-mediated folate uptake. Folate-labelled 125I-RNAase was found to bind to cells with high affinity (Kd = 24 nM), and like the free vitamin, its binding could be competitively blocked by excess free folate. Furthermore, binding could be reversed by either washing the cells with acid/saline, pH 3.0, or by treating the cells with phosphatidyl-inositol-specific phospholipase C, an enzyme known to release FBP from cell surfaces. Because cells pretreated with anti-FBP serum were unable to bind folate conjugates, and since the same antiserum identified a single 65 kDa band reminiscent of FBPs found in many other tissues, we conclude that a classical FBP is responsible for the uptake of folate-protein conjugates by receptor-bearing cells.
Rounding error, an unexpected fault in the output from a recording spectrophotometer: implications for model discrimination
Authors: Cárdenas M L , Cornish-Bowden A
Published: August 2015

Although commonly ignored in discussions of experimental error, rounding may sometimes be the major source of error, especially with modern precision instruments: some recording spectrophotometers are optically and photometrically capable of making absorbance measurements with errors less than 0.0003, but provide no numerical information more precise than +/- 0.001. The problem may be diagnosed by a characteristic arrangement of points in a residual plot, which resembles the result of cutting a stroboscopic picture of a bouncing ball into several strips and modifying it by sliding the strips relative to one another to bring the points closer to the axis. Harmful effects of rounding error can be critical in experiments designed for model discrimination.
The substance P fragment SP-(7-11) increases prostaglandin E2, intracellular Ca2+ and collagenase production in bovine articular chondrocytes
Authors: Halliday D A , McNeil J D , Betts W H , Scicchitano R
Published: August 2015

Substance P (SP) is found in increased concentrations in inflamed joints and is believed to play a role in joint pathology. Culture of bovine articular chondrocytes with SP or with the related mammalian tachykinins neurokinin A or B (NKA or NKB) produced no effect on prostaglandin E2 (PGE2) or collagenase production. However, the C-terminal fragment of SP, SP-(7-11), increased PGE2 and collagenase production at concentrations greater than 1 microM. The N-terminal fragments SP-(1-4) and SP-(1-6) had no effect on PGE2 or collagenase production. In addition, SP-(7-11), but not intact SP, SP-(1-4), SP-(1-6), SP-(8-11) or SP-(9-11), nor the tachykinins NKA and NKB, caused an increase in the intracellular calcium concentration as measured by the fluorescent dye Fura-2. The maximal change in intra-cellular calcium induced by 10 microM SP-(7-11) was 140 +/- 30 nM. We postulate that cleavage of SP by neutral endopeptidases which are present in the synovial fluid and which yield SP-(7-11) may be of biological importance in chondrocyte-mediated cartilage pathology.
Protein kinase C-dependent phosphorylation of annexins I and II in mesangial cells
Authors: Oudinet J P , Russo-Marie F , Cavadore J C , Rothhut B
Published: August 2015

In this study we describe the phosphorylation of annexins from cultured rat mesangial cells by protein kinase C (PKC) both in vitro and in vivo. Annexins I and II were detected either by Western-blot analysis or by immunoprecipitation using specific antibodies. In the presence of [gamma-32P]ATP, cytosolic annexin I and annexin II were phosphorylated in vitro only when Ca2+ and phospholipids were added, but not in the presence of phospholipids alone. Annexin I was shown to be a better substrate than annexin II. In experiments in vivo performed on 32P-labelled mesangial cells, the addition of two well-known activators of PKC, namely angiotensin II (AII) and phorbol myristate acetate (PMA), increased preferentially the phosphorylation of annexin I. Annexin II was phosphorylated to a much lesser extent after AII treatment. Phosphoamino acid analysis of annexins, either by two-dimensional chromatography or by using a specific antiphosphotyrosine antibody, revealed only phosphoserine in these experiments in vivo. The addition of AII to mesangial cells increased serine phosphorylation of annexin I and annexin II, whereas PMA only increased serine phosphorylation of annexin I. V8-protease phosphopeptide mapping of annexin I that was phosphorylated both in vitro and in vivo by PKC from mesangial cells shows similar phosphopeptides.
Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes
Authors: Bolt M J G , Bissonnette B M , Wali R K , Hartmann S C , Brasitus T A , Sitrin M D
Published: August 2015

The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5′-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5′-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5′-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.
The effects of elevated cyclic AMP levels on histamine-H1-receptor-stimulated inositol phospholipid hydrolysis and calcium mobilization in the smooth-muscle cell line DDT1MF-2
Authors: Dickenson J M , White T E , Hill S J
Published: August 2015

The effects of raising cyclic AMP levels, by forskolin stimulation, beta-adrenoceptor activation or cyclic AMP phosphodiesterase inhibition, on inositol phospholipid hydrolysis and increases in intracellular free [Ca2+] ([Ca2+]i) elicited by a range of agonists have been investigated in the hamster vas deferens smooth-muscle cell line DDT1MF-2. Isoprenaline (log [EC50 (M)] = -7.7 +/- 0.2), forskolin and the type IV cyclic AMP phosphodiesterase inhibitor rolipram elicited significant increases in the accumulation of cyclic [3H]AMP. Pretreatment with forskolin (10 microM) attenuated histamine (100 microM)- and N6-cyclopentyladenosine (CPA; 300 nM)-induced release of intracellular Ca2+, observed when cells are stimulated in Ca(2+)-free buffer containing 0.1 mM EGTA. Forskolin had no effect on ATP (100 microM)- or bradykinin (1 microM)-stimulated release of intracellular Ca2+. Histamine-induced intracellular Ca2+ release was also inhibited by pretreatment with rolipram (100 microM) or the membrane-permeant cyclic AMP analogue (Sp)-adenosine 3′,5′-monophosphothioate (100 microM). Isoprenaline (1 microM) pretreatment (in the presence of 10 microM rolipram, a concentration which on its own did not decrease the histamine response) attenuated histamine-induced intracellular Ca2+ release. Forskolin inhibited histamine (100 microM)- and CPA (100 nM) stimulated accumulation of [3H]-inositol phosphates, but was without effect on ATP or bradykinin responses. Addition of forskolin (in the presence of 100 microM rolipram) after the cells had been stimulated with histamine (in experiments initiated in Ca(2+)-free buffer) inhibited the rise in [Ca2+]i observed when extracellular Ca2+ (2 mM) was re-applied (owing to receptor-mediated Ca2+ influx). Finally, the refilling of intracellular Ca2+ stores (after receptor-mediated Ca2+ influx is blocked by mepyramine) can be demonstrated in the presence of raised cyclic AMP levels.
Prostaglandin E2 stimulates cyclic AMP-mediated hyaluronan synthesis in rabbit pericardial mesothelial cells
Authors: Honda A , Sekiguchi Y , Mori Y
Published: August 2015

We studied the effects of prostaglandin E2 (PGE2) on hyaluronan synthesis in rabbit pericardial mesothelial cells, and the following results were obtained. (1) PGE2 (10-1000 ng/ml) stimulated hyaluronan synthesis and the level of hyaluronan synthase activity in a dose- and time-dependent manner, but PGF2 alpha did not. (2) Cyclic AMP (cAMP) levels in the cells peaked (about a 7-fold increase) at 5-10 min after adding PGE2 (1000 ng/ml). (3) Increased hyaluronan synthesis induced by PGE2 was significantly inhibited after pretreatment with either an adenylate cyclase inhibitor (2′,5′-dideoxyadenosine) or a cAMP-dependent protein kinase inhibitor (PKI 5-24), but there was no inhibition with the protein kinase C inhibitor H-7. (4) When the intracellular cAMP level was raised by manipulating the levels of dibutyryl cyclic AMP or forskolin, hyaluronan synthesis and the level of hyaluronan synthase activity were also stimulated. These results suggest that PGE2 produced by cells stimulates hyaluronan synthesis in rabbit pericardial cells and that the stimulation mechanism involves the cAMP-mediated protein kinase signal transduction process.
Sub-second oscillations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate during platelet activation by ADP and thrombin: lack of correlation with calcium kinetics
Authors: Raha S , Jones G D , Gear A R L
Published: August 2015

The hypothesis that ADP and thrombin liberate Ins(1,4,5)P3 in blood platelets, with kinetics consistent for releasing Ca2+ within 2s, was tested by quenched-flow techniques. Both agonists stimulated transient and equal synthesis of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 near 200 ms and later short-lived peaks, which were not correlated with the slower steady increase in intracellular [Ca2+] between 0.5 to 2 s detected by Indo-1. Shear forces alone caused transient liberation of these inositol phosphates within 0.5 s and up to 4 s, yet failed to increase intracellular [Ca2+].
A class-A β-lactamase from Pseudomonas stutzeri that is highly active against monobactams and cefotaxime
Authors: Franceschini N , Galleni M , Frère J M , Oratore A , Amicosante G
Published: August 2015

A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation ‘beta-lactamase-stable’ cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes.
Regulation of fatty acid synthase gene transcription. Sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3-L1 preadipocytes are present in the 332 bp promoter
Authors: Moustaïd N , Sakamoto K , Clarke S , Beyer R S , Sul H S
Published: August 2015

We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5′-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5′ flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 +/- 0.2-, 2.6 +/- 0.1-, 2.0 +/- 0.2- and 1.7 +/- 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 +/- 0.3- and 3.5 +/- 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 +/- 0.8-, 3.0 +/- 0.7- and 2.3 +/- 0.5-fold respectively in serum-free media, and by 2.6 +/- 0.4-, 3.3 +/- 0.9-, 3.1 +/- 0.4- and 2.9 +/- 0.6-fold respectively in the presence of 0.5% serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.
Effects of inositol starvation on the levels of inositol phosphates and inositol lipids in Neurospora crassa
Authors: Lakin-Thomas P L
Published: August 2015

An inositol-requiring strain of Neurospora crassa was labelled during growth in liquid medium with [3H]inositol, and the levels of inositol phosphates and phosphoinositides were determined under inositol-sufficient and inositol-starved conditions. Because the mutant has an absolute requirement for inositol, the total mass of inositol-containing compounds could be determined. Inositol-containing lipids were identified by deacylation and co-migration with standards on h.p.l.c.; PtdIns3P, PtdIns4P, and PtdIns(4,5)P2 were found in approximately equal amounts, in addition to large amounts of PtdIns. Inositol starvation decreased the level of PtdIns to 10% of the sufficient level, and decreased the levels of the other phosphoinositides to about 25%. A number of inositol phosphates were found, including several InsP3s, InsP4s and InsP5s and phytic acid. Ins(1,4,5)P3 was identified by co-migration with standards on h.p.l.c. and by digestion with inositol phosphomonoesterase. High concentrations of all inositol phosphates were found in the extracellular medium in inositol-starved cultures. Inositol starvation on both liquid and solid agar media decreased the intracellular levels of some inositol phosphates, but increased the levels of phytic acid and several other inositol phosphates which may be its precursors and/or breakdown products. These results may indicate that inositol starvation induces phytic acid synthesis as a protection against the free-radical production and lipid peroxidation characteristic of inositol-less death.
Electron microscopy of cytochrome c oxidase-containing proteoliposomes: imaging analysis of protein orientation and monomer-dimer behaviour
Authors: Tihova M , Tattrie B , Nicholls P
Published: August 2015

1. Cytochrome c oxidase-containing vesicles were prepared by cholate dialysis using bovine heart cytochrome c oxidase with egg and dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamines (1:1, w/w) at two ratios of phospholipid to protein (25 mg/mg and 10 mg/mg). With each mixture, one or two (FII, FIII) fractions with mostly outward-facing cytochrome aa3 were separated from a fraction (FI) containing mostly inward-facing enzyme and protein-free liposomes by DEAE-Sephacel chromatography. 2. FII and FIII fractions from egg phospholipid mixtures had 60-80% outward-facing enzyme; FII and FIII fractions from dioleoyl phospholipids showed 50-70% outward-facing enzyme. Egg and dioleoyl phospholipid mixtures maintained good respiratory control ratios (8-13) only at the higher lipid/protein ratios. 3. Platinum/carbon replicas of freeze-fractured vesicle surfaces were subjected to image analysis. The results showed two types of membrane projection with average heights of 7.5 nm and 3.5 nm from the fracture plane. The former were more numerous on the convex faces. Calculated areas of the projections indicated the probable presence of both enzyme dimers and higher aggregates. Oxidase dimers may have membrane areas of 70-80 nm2 at the high (7.5 nm) side and 40-50 nm2 on the low (3.5 nm) side. 4. Proteoliposomes prepared with enzyme depleted of subunit III contained predominantly much smaller projecting areas. These probably represent monomers with high side areas of 35-40 nm2 and low side areas of 20-25 nm2. Electron microscopy thus directly confirms the predicted change of aggregation state resulting from subunit depletion. 5. The results are compared with those from two-dimensional crystals. Assuming that the high and low projections are two sides of one family of transmembrane molecules, a total length of 11 nm matches 11-12 nm lengths obtained by crystallography. Our membrane areas match the areas obtained in earlier ‘crystal’ studies better than the small areas obtained recently by electron cryomicroscopy.
Purification and partial characterization of lipoxygenase with dual catalytic activities from human term placenta
Authors: Joseph P , Srinivasan S N , Kulkarni A P
Published: August 2015

Lipoxygenase possessing dual catalytic activities, i.e. dioxygenase and hydroperoxidase, was purified from the cytosols of term placentas from non-smoking women. Concanavalin A affinity chromatography followed by phenyl-Sepharose CL-4B chromatography resulted in the separation of one hydrophobic and one non-hydrophobic isoenzyme. The concanavalin A-purified enzyme was used in all subsequent experiments. The dioxygenase activity of the enzyme exhibited a Vmax. of 204.37 +/- 17.66 nmol/min per mg of protein and a Km of 0.79 mM for linoleic acid. The involvement of dioxygen in enzymic linoleic acid oxidation was confirmed by O2 uptake studies. Arachidonic acid and linolenic acid also served as substrates for the dioxygenase activity. The placental lipoxygenase co-oxidized benzidine in the presence of linoleic acid (hydroperoxidase activity). Both the dioxygenase and hydroperoxidase activities were significantly stimulated by Ca2+ (1-100 microM), ATP (10-400 nM) and H2O2 (1-10 nM). Similarly, these two activities were inhibited by nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, gossypol, esculetin, butylated hydroxyanisole and butylated hydroxytoluene. Boiled enzyme was without significant dioxygenase and hydroperoxidase activities. Pyrogallol, 3,3′-dimethoxybenzidine, 3,3′,5,5′-tetramethylbenzidine, tetramethylphenylenediamine and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) were also co-oxidized by the placental lipoxygenase. These results suggest that: (i) lipoxygenase from human term placenta exhibits both dioxygenase and hydroperoxidase activities, and (ii) this enzyme represents an important pathway for chemical oxidation in the placentas of non-smoking women.
Activation of phospholipase A2 and stimulation of prostaglandin E2 production by transforming growth factor-α in rat thymic epithelial cells requires influx of calcium
Authors: Liu P , Wen M , Sun L , Hayashi J
Published: August 2015

The stimulation of both phospholipase A2 (PLA2) enzymic activity and the production of prostaglandin E2 (PGE2) by transforming growth factor-alpha (TGF-alpha) and Ca2+ ionophore A23187 in TEA3A1 rat thymic epithelial cells were studied. TGF-alpha by itself at various concentrations (5-200 ng/ml) had no effect on the stimulation of PGE2 production. A23187 (1 microgram/ml) by itself stimulated PGE2 production on average by 18-fold over the control. When TGF-alpha (50 ng/ml) was added to the cells in the presence of A23187, a synergistic stimulation (on average 45-fold) of PGE2 production was observed. Synergistic stimulation was also observed at the level of arachidonic acid released from phospholipid pools, suggesting the activation of PLA2 enzymic activity. We have found that this synergistic activation of PLA2 enzymic activity and subsequent stimulation of PGE2 production required the activation of epidermal growth factor (EGF) receptor tyrosine kinase and Ca2+ influx. This was shown by the fact that genistein, an inhibitor of tyrosine kinase, blocks the synergistic stimulation by TGF-alpha and A23187 and by the fact that the stimulation of PGE2 production by TGF-alpha and A23187 is dependent on the culture-medium Ca2+ concentrations. The requirement for Ca2+ influx instead of intracellular mobilization of Ca2+ was shown by the fact that PGE2 production was not stimulated when cells were treated with TGF-alpha and thapsigargin. Moreover, the synergistic stimulation of PGE2 production by TGF-alpha and A23187 was not affected in protein kinase C down-modulated cells. In addition, the synergistic stimulation was not observed in cells treated with either phorbol 12-myristate 13-acetate (PMA) and TGF-alpha or PMA and A23187, and in cells treated with TGF-alpha and thapsigargin. The requirement for the activation of receptor tyrosine kinase seems to be specific to the EGF receptor, since a synergistic stimulation of PGE2 production was not observed when cells are treated with either insulin-like growth factor-I or fibroblast growth factor-I in the presence of A23187.
Rapid purification and direct microassay of calbindin9kDa utilizing its solubility in perchloric acid
Authors: Hubbard M J
Published: August 2015

The 9 kDa calcium-binding protein, calbindin9kDa, was found to be soluble in 7% (v/v) perchloric acid. Calbindin9kDa was easily purified from rat duodenum in 1 day with perchloric acid precipitation followed by reverse-phase h.p.l.c. The yield was 21.4 +/- 2.3 nmol/g wet weight of tissue (mean +/- S.E.M.; n = 3) from normally fed 7-8-week-old rats (approx. 70% recovery). The purification was also effective with rabbit duodenum calbindin9kDa, but not with various other EF-hand calcium-binding proteins tested in the rat. Several criteria (h.p.l.c., u.v. spectrum, denaturing two-dimensional PAGE, N-terminal sequencing) indicated that the rat calbindin9kDa was purified to homogeneity and was not affected by proteolysis. High-affinity calcium-binding properties were retained and no evidence of isoforms or charge modification was observed. Residue 59, identified as Asn (not Asp as previously reported), was fully amidated. When adopted as a microassay with isocratic h.p.l.c., the perchloric acid procedure enabled rapid (less than 6 min) and direct (peptide bond absorbance) quantification of less than 1 pmol of calbindin9kDa. This new approach to purification and assay will be of particular utility for investigations of calbindin9kDa in previously intractable low-abundance sources (e.g. cultured cells).
Influence of Mg2+ on detection of somatogenic and lactogenic components of growth-hormone-binding protein in mammalian sera
Authors: Amit T , Hochberg Z , Barkey R J
Published: August 2015

We recently classified the growth-hormone (GH)-binding protein (GH-BP) in a wide range of mammalian [including human (h)] sera and reported the existence of a major lactogenic component in GH-BP of type-III sera (rabbit, horse, dog, pig and cat), based on the capacity of bovine (b) and ovine prolactin (PRL) to displace 125I-labelled human growth hormone (hGH) binding and on direct 125I-bPRL binding studies. In this study, we demonstrate the high degree of Mg2+ dependence of the binding of the classically lactogenic hGH and bPRL, but not that of the somatogenic bGH to various mammalian sera (types I-IV). Serum GH-BP was assayed using a previously described and validated charcoal-separation assay. 125I-hGH binding to rat, ovine, bovine, rabbit, horse, dog and human sera was enhanced 1.5-2.5-fold in the presence of 70 mM Mg2+. The Mg2+ effect was concentration-dependent between 3.7 mM and 70 mM, causing a significant and proportional increase in 125I-hGH binding to serum. Like 125I-hGH, 125I-bPRL binding to type-III sera was also Mg(2+)-dependent. In contrast, 125I-bGH binding to all types of serum GH-BP was not affected by Mg2+ concentrations of up to 35 mM, while 70 mM Mg2+ slightly, but significantly, reduced (by approx. 15%) bGH binding to rabbit serum. In keeping with the Mg(2+)-dependent stimulation of lactogenic hormone binding to GH-BP, 70 mM Mg2+ caused a shift to the left in the displacement curves of hGH and bPRL competing with 125I-hGH binding to rabbit, dog, horse and human sera, while the effects of the somatogens bGH and rabbit GH were shifted to the right. Scatchard analysis of hGH displacement curves with sera from various species yielded linear plots and revealed that Mg2+ significantly increased (2.3-3.0-fold) the affinity constants, but not the binding capacities. These results demonstrate the ability of changes in Mg2+ concentration to determine the degree of differential recognition of somatogens versus lactogens by serum GH-BP. It remains to be determined whether such bivalent cation effects may account, at least in part, for the growth retardation seen in Zn2+ or Mg2+ ion deficiencies.
Calcium measurements with a new high-affinity n.m.r. indicator in the isolated perfused heart
Authors: Kirschenlohr H L , Grace A A , Clarke S D , Shachar-Hill Y , Metcalfe J C , Morris P G , Smith G A
Published: August 2015

A new n.m.r. indicator, 1,2-bis-(2-[1-(hydroxycarbony)ethyl- (hydroxycarbonylmethyl)]amino-5-fluorophenoxy)ethane (DiMe-5FBAPTA), with a higher affinity for calcium (apparent Kd 46 nM, pH 7.2, 30 degrees C) than the parent 5FBAPTA chelator (Kd 537 nM, pH 7.1, 30 degrees C) has been used to measure the cardiac intracellular free Ca2+ ([Ca2+]i). DiMe-5FBAPTA was loaded into Langendorff-perfused ferret hearts maintained at 30 degrees C using the acetoxymethyl ester (AM) derivative. The intracellular concentration required to achieve an adequate signal-to-noise (S/N) ratio (> 10:1) for the n.m.r. spectra caused a similar reduction in developed pressure to that obtained using 5FBAPTA-AM. The DiMe-5FBAPTA was used to estimate [Ca2+]i in diastole, through the calcium transient and at rest in the presence of the slow calcium channel blocker diltiazem. At a pacing frequency of 1.0 Hz, end-diastolic [Ca2+]i was 198 +/- 30 nM (n = 9), and reducing the pacing frequency to 0.2 Hz lowered [Ca2+]i to 89 +/- 13 nM (n = 5). Perfusion with diltiazem (100 microM) for 60 min lowered [Ca2+]i to 10 +/- 1 nM (n = 4) in unpaced hearts and to 94 +/- 24 nM (n = 4) in hearts paced at 1.0 Hz. The [Ca2+]i transient measured with DiMe-5FBAPTA was sharper and delayed compared with the transient measured previously with 5FBAPTA. Co-loading the two indicators provided evidence that the indicator with the higher Kd had a dominant effect on the end-diastolic [Ca2+]i. The lower values for end-diastolic [Ca2+]i obtained with DiMe-5FBAPTA are consistent with fluorescent indicator measurements. These observations suggest that perturbations of [Ca2+]i caused by the new indicator are less than those induced by 5FBAPTA. DiMe-5FBAPTA therefore represents a useful step in the development of 19F-n.m.r. calcium indicators.
Multiple mechanisms by which protein kinase A potentiates inositol 1,4,5-trisphosphate-induced Ca2+ mobilization in permeabilized hepatocytes
Authors: Hajnóczky G , Gao E , Nomura T , Hoek J B , Thomas A P
Published: August 2015

The mobilization of Ca2+ from intracellular stores by Ins(1,4,5)P3 in suspensions of permeabilized rat hepatocytes was potentiated by preincubating intact cells with adenosine 3′:5′-cyclic phosphorothioate (cpt-cAMP), or by addition of the catalytic subunit of cyclic-AMP-dependent protein kinase (PKA) after cell permeabilization. This action of PKA involved both an enhancement in Ins(1,4,5)P3 sensitivity and an increase in the size of the Ins(1,4,5)P3-releasable Ca2+ pool. Inclusion of the protein phosphatase inhibitor okadaic acid in the permeabilization medium augmented the effects of PKA. Treatment with PKA catalytic subunit also increased the rate of ATP-dependent Ca2+ sequestration. To determine whether the effects of PKA on the Ca(2+)-release mechanism were secondary to alterations in the Ca2+ load of the Ins(1,4,5)P3-sensitive stores, a method was developed using Mn2+ as a Ca2+ surrogate to examine the permeability properties of the Ins(1,4,5)P3-gated channels independent of Ca2+ fluxes. This approach utilized the ability of Mn2+ to quench the fluorescence of fura-2 compartmentalized within intracellular Ca2+ stores in an Ins(1,4,5)P3-dependent manner, with thapsigargin added to block the ATP-activated Ca2+ pump and to ensure that the Ca2+ stores were fully depleted of Ca2+. The initial rate and extent of Mn2+ quenching of compartmentalized fura-2 was increased in a dose-dependent manner by Ins(1,4,5)P3. PKA activation increased both the initial rate and the extent of Mn2+ quenching at sub-maximal Ins(1,4,5)P3 doses, but there was no effect on the quench rate in the presence of saturating Ins(1,4,5)P3. However, the amount of compartmentalized fura-2 that could be quenched by Mn2+ in the presence of maximal Ins(1,4,5)P3 was increased by PKA. These data suggest two distinct actions of PKA on the Ins(1,4,5)P3-sensitive Ca2+ stores. (1) Modification of the ion-permeability properties of the Ins(1,4,5)P3 receptor/channel through an increase in the sensitivity to Ins(1,4,5)P3 for channel opening. (2) A recruitment of Ca2+ stores from the Ins(1,4,5)P3-insensitive pool. Both actions were independent of the Ca(2+)-loading state of the stores. Imaging studies of single permeabilized hepatocytes showed that the Ins(1,4,5)P3-sensitive stores were distributed throughout the cell and PKA enhanced the rate of Ins(1,4,5)P3-stimulated Mn2+ quench in individual cells, without modifying the subcellular distribution of Ins(1,4,5)P3-sensitive stores.
Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself
Authors: Mach L , Schwihla H , Stüwe K , Rowan A D , Mort J S , Glössl J
Published: August 2015

In order to elucidate the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent autoprocessing. When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed. Inhibitors of metallo-, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro. However, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074. Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B. On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself.
Inactivation of the endogenous argininosuccinate lyase activity of duck δ-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate
Authors: Lee H J , Chiou S H , Chang G G
Published: August 2015

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.
Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress
Authors: Petronini P G , De Angelis E M , Borghetti A F , Wheeler K P
Published: August 2015

Induced expression of the HSP70 gene in 3T3 and SV-3T3 cells was monitored by measurements of the synthesis of HSP70 and of the cellular contents of both HSP70 and its mRNA. The presence of betaine (N-trimethylglycine) at concentrations of 2.5-25 mM decreased the induction of HSP70 gene expression caused by incubation of 3T3 and SV-3T3 cells in hypertonic (0.5 osM) medium. This effect was accompanied by an enhancement of SV-3T3 cell adaptation, assayed by colony formation, to the hyperosmotic conditions. In contrast, the presence of betaine did not affect HSP70 gene expression induced in these cells by heat shock. After 6 h incubation with 25 mM betaine under hypertonic (0.5 osM) conditions the intracellular concentration of betaine in SV-3T3 cells was about 195 mM, compared with about 70 mM under isotonic (0.3 osM) conditions. Hence, with this concentration of extracellular betaine, the marked increase in the accumulation of betaine within the cells presumably counteracts the imposed osmotic pressure and eliminates the signal that otherwise initiates increased expression of the HSP70 gene.
Hepatocyte heterogeneity in response to extracellular adenosine
Authors: Morimoto Y , Wettstein M , Häussinger D
Published: August 2015

Metabolic and haemodynamic effects of adenosine were studied in antegrade and retrograde rat liver perfusions with influent nucleoside concentrations either below (i.e. 20 microM) or exceeding (i.e. 200-300 microM) the single-pass clearance capacity of the liver. Adenosine (20 microM) increased in antegrade perfusions the perfusion pressure and markedly stimulated prostaglandin D2, thromboxane B2 and glucose output, whereas in retrograde perfusions no pressure and eicosanoid response occurred and glucose output was stimulated only slightly. The perfusion-direction-dependent differences in the glucose and pressure response to adenosine (20 microM) were fully abolished in presence of ibuprofen (50 microM). When the adenosine concentration in influent was raised to 200-300 microM, i.e. to a concentration exceeding single-pass clearance of the nucleoside, the adenosine-induced prostaglandin D2 release was about 10-fold higher in retrograde perfusions than in antegrade perfusions. On the other hand, both adenosine (20-300 microM)-induced cyclic AMP (cAMP) and K+ release from the liver were not affected by the direction of perfusion, and maximal effects on cAMP release were observed at influent adenosine concentrations of 100 microM. The basal rate (adenosine absent) of prostaglandin D2 and thromboxane B2 release was about 10-fold higher in retrograde than in antegrade perfusion experiments, whereas the basal cAMP release from the liver was not affected by the direction of perfusion. Maximal adenosine-stimulated glucose output was significantly higher in antegrade than in retrograde perfusions at all adenosine concentrations tested (range 10-300 microM). Ibuprofen abolished this difference, indicating that eicosanoids liberated under the influence of adenosine contribute to the glycogenolytic response in antegrade, but not in retrograde, perfusion. Desensitization occurred following repetitive adenosine infusion; this was more pronounced for adenosine-induced prostaglandin release than for cAMP or K+ efflux. The data suggest the following. (i) Both cAMP and eicosanoids are involved in the stimulation of glycogenolysis by adenosine. (ii) Eicosanoids are probably liberated under the influence of extracellular adenosine from a portal pre-sinusoidal compartment and accordingly stimulate glycogenolysis only in antegrade perfusions. Thus signals derived from portal vein structures can modulate hepatocellular function. (iii) Contractile elements are probably located also inside the liver acinus. (iv) Eicosanoids released into the hepatic vein reflect less than 10% of hepatic eicosanoid formation, because of marked clearance by perivenous hepatocytes.
Interactions between active-site-serine β-lactamases and compounds bearing a methoxy side chain on the α-face of the β-lactam ring: kinetic and molecular modelling studies
Authors: Matagne A , Lamotte-Brasseur J , Dive G , Knox J R , Frère J M
Published: August 2015

The interactions between three class A beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring (cefoxitin, moxalactam and temocillin) have been studied. When compared with the situation prevailing with good substrates, both acylation and deacylation steps appeared to be severely impaired. Molecular modelling studies of the structures of the Henri-Michaelis complexes and of the acyl-enzymes indicate a major displacement of the crystallographically observed water molecule which connects the glutamate-166 and serine-70 side chains and underline the role of this water molecule in both reaction steps.
Developmental and biochemical characteristics of the cardiac membrane-bound arginine-specific mono-ADP-ribosyltransferase
Authors: McMahon K K , Piron K J , Ha V T , Fullerton A T
Published: August 2015

ADP-ribosylation of protein in heart membrane preparations has been shown to be present in adult tissue but absent from early neonate tissue [Piron and McMahon (1990) Biochem. J. 270, 591-597]. To further this observation, the cardiac membrane-bound form of arginine-specific mono-ADP-ribosyltransferase (EC 2.4.2.31) has been characterized. Apparent Km values of 330 and 470 microM were found in heart membrane preparations from rat and quail respectively. The Vmax. value depended greatly on the species of animal studied, and was 1.1 and 48 nmol/min per mg in rat and quail preparations respectively. The specific activity of the enzyme was lowest in pig, intermediate in rat, dog and rabbit, and highest in mouse and quail cardiac membranes. In the rat, the ADP-ribosylation of protein and enzyme activity were very low in heart preparations from 1-15-day-old animals. Thereafter the ADP-ribosylation and enzyme activity increased gradually to adulthood. Bacillus cereus phosphatidylinositol-specific phospholipase C, known to hydrolyse glycosylphosphatidylinositol anchors of proteins, released the mono-ADP-ribosyltransferase from membrane preparations of both rat and quail in a dose-dependent, Zn(2+)-inhibited manner. Thus it appears that a membrane-bound form of arginine-specific mono-ADP-ribosyltransferase is present in heart membranes from a variety of species and is not species-specific. The activity of this ADP-ribosyltransferase appears to be developmentally regulated and to be bound to the cardiac membranes by a glycosylphosphatidylinositol anchor.