bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet phospholipase D is activated by protein kinase C via an integrin αIIbβ3-independent mechanism
Authors: Martinson E A , Scheible S , Greinacher A , Presek P
Published: August 2015

Blood platelets contain phospholipase D (PLD) that is rapidly activated following platelet stimulation. It is currently unclear, however, where PLD fits into the signalling cascade that leads to aggregation and secretion. Therefore we investigated the mechanism of activation of PLD in human platelets, using the formation of the PLD-specific product phosphatidylethanol as a measure of PLD activity. PLD was activated by a number of platelet agonists that also cause the activation of protein kinase C, including thrombin, collagen, the Ca2+ ionophore A23187 and the thromboxane A2-mimetic U46619. Phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C, also increased PLD activity. A selective inhibitor of protein kinase C, Ro-31-8220, totally blocked the stimulation of PLD by thrombin or PMA under conditions in which it also inhibited phosphorylation of pleckstrin, the major protein kinase C substrate in platelets. Ro-31-8220 additionally inhibited A23187-stimulated PLD activity, indicating that Ca2+ activation of PLD also occurs via a protein kinase C-dependent pathway. In the presence of the fibrinogen antagonist peptide RGDS, which inhibits fibrinogen binding to integrin alpha IIb beta 3 and allows little or no aggregation to occur, thrombin- and PMA-stimulated PLD activity was still observed, indicating that PLD activation is not simply a consequence of platelet aggregation. Furthermore, these agonists were able to stimulate PLD in platelets from a Glanzmann's thrombasthenia type I patient lacking the integrin alpha IIb beta 3 complex, which indicates that activation of PLD is also independent of the recruitment of integrin alpha IIb beta 3. Taken together, our results show that PLD is activated by a pathway involving protein kinase C, and suggest that PLD might be involved in signal transduction events occurring upstream of integrin alpha IIb beta 3 activation and fibrinogen binding, which are prerequisites for full platelet aggregation.
An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system
Authors: de Vries K J , Heinrichs A A J , Cunningham E , Brunink F , Westerman J , Somerharju P J , Cockcroft S , Wirtz K W A , Snoek G T
Published: August 2015

An isoform of the phosphatidylinositol-transfer protein (PI-TP) was identified in the cytosol fraction of bovine brain. This protein, designated PI-TP beta, has an apparent molecular mass of 36 kDa and an isoelectric point of 5.4. The N-terminal amino acid sequence (21 residues) is 90% similar to that of bovine brain PI-TP, henceforth designated PI-TP alpha (molecular mass 35 kDa and pI 5.5). As observed for PI-TP alpha, PI-TP beta has a distinct preference for phosphatidylinositol over phosphatidylcholine. In addition, it expresses a high transfer activity towards sphingomyelin. PI-TP alpha lacks this activity completely. By indirect immunofluorescence we demonstrated that, in Swiss mouse 3T3 fibroblasts, PI-TP beta is preferentially associated with the Golgi system whereas PI-TP alpha is predominantly present in the cytoplasm and the nucleus. In cytosol-depleted HL60 cells, both PI-TP alpha and PI-TP beta were equally effective at reconstituting guanosine 5′-[gamma-thio]triphosphate-mediated phospholipase C beta activity.
Functions of the C-terminal domain of CTP: phosphocholine cytidylyltransferase. Effects of C-terminal deletions on enzyme activity, intracellular localization and phosphorylation potential
Authors: Cornell R B , Kalmar G B , Kay R J , Johnson M A , Sanghera J S , Pelech S L
Published: August 2015

The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II, cell division control 2 kinase (cdc2 kinase), protein kinases C alpha and beta II, and glycogen synthase kinase-3 (GSK-3), but not by mitogen-activated kinase (MAP kinase). Casein kinase II phosphorylation was directed exclusively to Ser-362. The sites phosphorylated by cdc2 kinase and GSK-3 were restricted to several serines within three proline-rich motifs of domain P. Sites phosphorylated in vitro by protein kinase C, on the other hand, were distributed over the N-terminal catalytic as well as the C-terminal regulatory domain. The stoichiometry of phosphorylation catalysed by any of these kinases was less than 0.2 mol P/mol CT, and no effects on enzyme activity were detected. This study supports a tripartite structure for CT with an N-terminal catalytic domain and a C-terminal regulatory domain comprised of a membrane-binding domain (domain M) and a phosphorylation domain (domain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.
Sequence requirements for proinsulin processing at the B-chain/C-peptide junction
Authors: Kaufmann J E , Irminger J C , Halban P A
Published: August 2015

Proinsulin is converted into insulin by the action of two endoproteases. Type I (PC1/PC3) is thought to cleave between the B-chain and the connecting peptide (C-peptide) and type II (PC2) between the C-peptide and the A-chain. An acidic region immediately C-terminal to the point of cleavage at the B-chain/C-peptide junction is well conserved throughout evolution and has been suggested to be important for proinsulin conversion [Gross, Villa-Komaroff, Kahn, Weir and Halban (1989) J. Biol. Chem. 264, 21486-21490]. We have here compared the precise role of this region as a whole and just the first acidic residue C-terminal to the point of cleavage in processing of proinsulin by PC3. To this end, several mutations were introduced in this region of human proinsulin (native sequence, B-chain RREAEDL C-peptide): RRPAEDL (C1Pro mutant); RRLAEDL (C1Leu mutant); RRL (C1-C4del mutant); RRE (del-C1Glu mutant). Mutant and native cDNAs were stably transfected into AtT20 (pituitary corticotroph) cells, in which PC3 is known to be the major conversion endoprotease, and kinetics of proinsulin conversion were studied (pulse-chase/HPLC analysis of proinsulin-related peptides). The results show that the acidic region following the B-chain/C-peptide junction is indeed important for PC3 cleavage at this site, and that the reduced cleavage observed for the C1-C4del mutant proinsulin can be partially overcome by replacing the acidic region with a single acidic residue (del-C1Glu mutant). Replacing only the first residue of the acidic region with leucine (C1Leu mutant) has no impact on conversion, whereas its replacement with proline (C1Pro mutant) almost completely abolishes cleavage at the B-chain/C-peptide junction without affecting that at the C-peptide/A-chain junction.
Effect of replacement of ferriprotoporphyrin IX in the haem domain of cytochrome P-450 BM-3 on substrate binding and catalytic activity
Authors: Modi S , Primrose W U , Lian L Y , Roberts G C K
Published: August 2015

Bacillus megaterium cytochrome P-450 BM-3 (coded by gene CYP102) is a catalytically self-sufficient mono-oxygenase, with both cytochrome P-450 and NADPH:cytochrome P-450 reductase domains, that catalyses the hydroxylation of fatty acids. The natural ferriprotoporphyrin IX has been removed from the haem domain of cytochrome P-450 BM-3 by treatment with acidified acetone, and it has been shown that, under carefully controlled conditions, haem can be added back to the resultant apoprotein to obtain a fully reconstituted haem domain with spectroscopic, substrate-binding and catalytic properties indistinguishable from those of the native domain. Replacement of the natural haem with ferriprotoporphyrin IX dimethyl ester yields a protein which has a higher affinity for the substrate dodecanoic acid and (in the presence of the reductase domain) the same catalytic rate as the native haem domain. Replacement with ferrimesoporphyrin IX yields a protein with the same affinity for substrate, but a reduced catalytic turnover. These results suggest that the haem moiety has a role in the creation of the binding pocket for substrate, and that modification of the electron density on the haem iron effects the catalytic rate.
Mutations of the basic amino acid transporter gene associated with cystinuria
Authors: Miyamoto K , Katai K , Tatsumi S , Sone K , Segawa H , Yamamoto H , Taketani Y , Takada K , Morita K , Kanayama H , Kagawa S , Takeda E
Published: August 2015

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.
Endothelin-induced changes in intracellular pH and Ca2+ in coronary smooth muscle: role of Na+-H+ exchange
Authors: Hubel C A , Highsmith R F
Published: August 2015

The relationship between endothelin-1 (ET-1)-induced stimulation of Na(+)-H+ exchange and intracellular free Ca2+ ([Ca2+]i) was examined in primary cultures of porcine coronary artery smooth muscle cells. Intracellular pH (pHi) and [Ca2+]i were measured using 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein and the acetoxymethyl ester of fura-2 respectively. In HCO3(-)-free buffer (pH = 7.4), ET-1 (0.1-50 nM) induced a sustained, dose-dependent increase in pHi. ET-1 (10 nM) increased pHi from 6.83 +/- 0.01 to 6.93 +/- 0.02 (P < 0.01). The alkalinization was blocked by the Na(+)-H+ exchange inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 3 microM) or by substitution of Na+ with N-methylglucamine or choline chloride (P < 0.05). Recovery of pHi in response to acidification, induced by washout of a 20 mM NH4Cl prepulse, was > 90% inhibited by EIPA (3 microM), confirming the presence of an ET-1-responsive Na(+)-H+ exchanger. Coronary smooth muscle cells responded to ET-1 with a dose-dependent, biphasic increase in [Ca2+]i which was not inhibited by manipulations (EIPA pretreatment or Na(+)-free media) shown to block the Na(+)-H+ exchanger. The ET-1-mediated alkalinization was not inhibited by removal of extracellular Ca2+ ([Ca2+]o). However, complete blockade of the ET-1-mediated [Ca2+]i response using the intracellular Ca(2+)-chelator, [bis-(2-amino-5-methylphenoxy)ethane-NNN'N′-tetraacetic acid tetraacetoxymethyl ester] (MAPTAM), in [Ca2+]o-free media, demonstrated that an increment in [Ca2+]i is required for activation of the Na(+)-H+ exchanger by ET-1. The ET-1-induced rise in [Ca2+]i was not associated with a rise in pHi in the presence of CO2/HCO3-. We conclude that: (1) activation of Na(+)-H+ exchange by ET-1 requires an increment in [Ca2+]i; and (2) ET-1 stimulates EIPA-sensitive Na(+)-H+ exchange, but this stimulation does not modulate ET-1-induced changes in [Ca2+]i.
Cloning and analysis of the promoter region of the rat SM22α gene
Authors: Kemp P R , Osbourn J K , Grainger D J , Metcalfe J C
Published: August 2015

We have cloned and sequenced a 1.9 kb fragment of the 5′-upstream sequence of the smooth-muscle-specific gene SM22 alpha. The region cloned consisted of the SM22 alpha promoter, a 65 bp exon containing most of the 5′-untranslated region and 307 bp of the first intron. A 1.5 kb fragment at the 5′ end of this sequence was able to drive the expression of a reporter chloramphenicol acetyltransferase (CAT) gene in both vascular smooth-muscle cells and Rat-1 fibroblasts. This promoter region did not contain a consensus TATAA box but contained the sequence TTTAAA 25 bp from the major start site identified by primer extension. Deletion analysis showed that a fragment of the promoter from +65 to -303 was more active in both cell types than the 1.5 kb fragment suggesting that there are silencer sequences in the region 5′ to the core promoter. CAT activity was also observed with fragments containing bases +65 to -193 and +65 to -117 in smooth-muscle cells. In contrast with the smooth-muscle cells, no CAT activity was detected in Rat-1 fibroblasts with the smallest two fragments. The residual promoter activity in the smallest fragment of the SM22 alpha promoter tested suggested that, unlike the smooth-muscle alpha-actin promoter, transcription from the SM22 alpha promoter can occur in smooth-muscle cells in the absence of factors binding to CC(A/Trich)6GG (CArG box) or CANNTG (E box) motifs.
Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi
Authors: Shen H , Gilkes N R , Kilburn D G , Miller R C , Warren R A J
Published: August 2015

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.
Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?
Authors: Heyworth P G , Erickson R W , Ding J , Curnutte J T , Badwey J A
Published: August 2015

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases.
Failure of insulin-regulated recruitment of the glucose transporter GLUT4 in cardiac muscle of obese Zucker rats is associated with alterations of small-molecular-mass GTP-binding proteins
Authors: Uphues I , Kolter T , Goud B , Eckel J
Published: August 2015

Cardiac ventricular tissue of lean and genetically obese (fa/fa) Zucker rats was used to study the expression, subcellular distribution and insulin-induced recruitment of the glucose transporter GLUT4 and to elucidate possible molecular alterations of the translocation process. Hearts were removed from basal and insulin-treated (20 min) lean and obese Zucker rats, and processed for subcellular fractionation and Western blotting of proteins. In obese rats, the total GLUT4 content in a crude membrane fraction was reduced to 75 +/- 8% (P = 0.019) of lean controls. In contrast, GLUT4 abundance in plasma membranes was not significantly different between lean and obese rats with a concomitant decrease (47 +/- 3%) in the microsomal fraction of obese animals. In plasma membranes of lean animals insulin was found to increase the GLUT4 abundance to 294 +/- 43% of control with a significantly (P = 0.009) reduced effect in the obese group (139 +/- 10% of control). In these animals insulin failed to recruit GLUT4 from the microsomal fraction, whereas the hormone induced a significant decrease (41 +/- 4%) of microsomal GLUT4 in lean controls. In GLUT4-enriched membrane vesicles, obtained from cardiac microsomes of lean rats, a 24 kDa GTP-binding protein could be detected, whereas no significant labelling of this species was observed in GLUT4 vesicles prepared from obese animals. In addition to the translocation of GLUT4, insulin was found to promote the movement of the small GTP-binding protein rab4A from the cytosol (decrease to 61 +/- 13% of control) to the plasma membrane (increase to 177 +/- 19% of control) in lean rats with no effect of the hormone on rab4A redistribution in the obese group. In conclusion, cardiac glucose uptake of insulin-resistant obese Zucker rats is subject to multiple cellular abnormalities involving a reduced expression, altered redistribution and defective recruitment of GLUT4. We show here an association of the latter defect with alterations at the level of small GTP-binding proteins possibly leading to an impaired trafficking of GLUT4 in the insulin-resistant state.
Modulation of hepatic apolipoprotein B, 3-hydroxy-3-methylglutaryl-CoA reductase and low-density lipoprotein receptor mRNA and plasma lipoprotein concentrations by defined dietary fats. Comparison of trimyristin, tripalmitin, tristearin and triolein
Authors: Bennett A J , Billett M A , Salter A M , Mangiapane E H , Bruce J S , Anderton K L , Marenah C B , Lawson N , White D A
Published: August 2015

Different dietary fatty acids exert specific effects on plasma lipids but the mechanism by which this occurs is unknown. Hamsters were fed on low-cholesterol diets containing triacylglycerols enriched in specific saturated fatty acids, and effects on plasma lipids and the expression of genes involved in hepatic lipoprotein metabolism were measured. Trimyristin and tripalmitin caused significant rises in low-density lipoprotein (LDL) cholesterol which were accompanied by significant reductions in hepatic LDL receptor mRNA levels. Tripalmitin also increased hepatic expression of the apolipoprotein B gene, implying an increased production of LDL via very-low-density lipoprotein (VLDL) and decreased removal of LDL in animals fed this fat. Hepatic levels of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not vary significantly between the groups. Compared with triolein, tristearin had little effect on hepatic gene expression or total plasma cholesterol. However, it caused a marked decrease in VLDL cholesterol and a rise in LDL cholesterol such that overall it appeared to be neutral. Lipid analysis suggested a rapid desaturation of much of the dietary stearate. The differential changes in plasma lipids and hepatic mRNA levels induced by specific dietary fats suggests a role for fatty acids or a metabolite thereof in the regulation of the expression of genes involved in lipoprotein metabolism.
Defective signal-transduction pathways in T-cells from autoimmune MRL-lpr/lpr mice are associated with increased polyamine concentrations
Authors: Thomas T J , Gunnia U B , Seibold J R , Thomas T
Published: August 2015

We previously reported that difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, exerted significant beneficial effects on the lifespan and disease expression of MRL-lpr/lpr mice, which spontaneously develop a lupus-like syndrome. Polyamine levels in splenic T-cells of MRL-lpr/lpr mice were significantly higher than those of Balb/c mice. In the present investigation, we examined the role of endogenous polyamines in transmembrane Ca2+ influx, generation of InsP3 and tyrosine phosphorylation of the p56lck protein in concanavalin A-stimulated splenic T-cells. Cytosolic free calcium concentrations ([Ca2+]i) in concanavalin A-stimulated T-cells of MRL-lpr/lpr and Balb/c mice were 250 +/- 25 and 450 +/- 42 nM respectively. Treatment of MRL-lpr/lpr mice with DFMO increased [Ca2+]i to 360 +/- 30 nM (P < 0.05). InsP3 levels of concanavalin A-stimulated MRL-lpr/lpr splenic T-cells were only 20% higher than those of unstimulated controls, whereas those of Balb/c T-cells were 90% higher. DFMO treatment increased InsP3 levels in concanavalin A-treated MRL-lpr/lpr T-cells to 67%. Western-blot analysis showed a 7-fold higher level of p56lck phosphorylation of MRL-lpr/lpr splenic T-cells than that of Balb/c mice. DFMO treatment reduced tyrosine phosphorylation of p56lck of MRL-lpr/lpr mice significantly (P < 0.001). Two-colour flow-cytometric analysis revealed no significant difference in the CD4+/CD8+ ratio in splenic T-cells of MRL-lpr/lpr mice after DFMO treatment. Polyamine levels in splenocytes were significantly reduced by DFMO treatment. These data show that DFMO treatment could alter signal-transduction pathways of splenic T-cells of MRL-lpr/lpr mice. Increased levels of polyamines in T-cells of untreated lpr mice contribute to defective signal-transduction pathways and the pathogenesis of lupus-like symptoms.
Muscarinic stimulation exerts both stimulatory and inhibitory effects on the concentration of cytoplasmic Ca2+ in the electrically excitable pancreatic B-cell
Authors: Gilon P , Nenquin M , Henquin J C
Published: August 2015

Mouse pancreatic islets were used to investigate how muscarinic stimulation influences the cytoplasmic Ca2+ concentration ([Ca2+]i) in insulin-secreting B-cells. In the absence of extracellular Ca2+, acetylcholine (ACh) triggered a transient, concentration-dependent and thapsigargin-inhibited increase in [Ca2+]i. In the presence of extracellular Ca2+ and 15 mM glucose, ACh induced a biphasic rise in [Ca2+]i. The initial, transient phase increased with the concentration of ACh, whereas the second, sustained, phase was higher at low (0.1-1 microM) than at high (> or = 10 microM) concentrations of ACh. Thapsigargin attenuated (did not suppress) the first phase of the [Ca2+]i rise and did not affect the sustained response. This sustained rise was inhibited by omission of extracellular Na+ (which prevents the depolarizing action of ACh) and by D600 or diazoxide (which prevent activation of voltage-dependent Ca2+ channels). During steady-state stimulation, the Ca2+ action potentials in B-cells were stimulated by 1 microM ACh but inhibited by 100 microM ACh. When B-cells were depolarized by 45 mM K+, ACh induced a concentration-dependent, biphasic change in [Ca2+]i, consisting of a first peak rapidly followed by a decrease. Thapsigargin suppressed the peak without affecting the drop in [Ca2+]i. Measurements of 45Ca2+ efflux under similar conditions indicated that ACh decreases Ca2+ influx and slightly increases the efflux. All effects of ACh were blocked by atropine. In conclusion, three mechanisms at least are involved in the biphasic change in [Ca2+]i that muscarinic stimulation exerts in excitable pancreatic B-cells. A mobilization of Ca2+ from the endoplasmic reticulum contributes significantly to the first peak, but little to the steady-state rise in [Ca2+]i. This second phase results from an influx of Ca2+ through voltage-dependent Ca2+ channels activated by a Na(+)-dependent depolarization. However, when high concentrations of ACh are used, Ca2+ influx is attenuated.
Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells
Authors: Anderson L , Alexander C L , Faccenda E , Eidne K A
Published: August 2015

This study uses fluorescence microscopy combined with dynamic video imaging to examine the events associated with the rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R). In single non-pituitary human embryonic kidney 293 (HEK-293) cells, expressing either the rat or human TRH-Rs, TRH produced a rapid dose-dependent monophasic rise in [Ca2+]i. This Ca2+ transient was completely abolished by pretreatment of cells with the intracellular Ca2+ antagonists thapsigargin or cyclopiazonic acid, but not EGTA, the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine or the second-messenger-operated Ca2+ channel antagonist SK&F 96365. These results suggest that TRH causes the mobilization of Ca2+ from thapsigargin/cyclopiazonic acid-sensitive intracellular Ca2+ stores but not the influx of extracellular Ca2+. HEK-293 cells also failed to respond to KCl or the slow Ca(2+)-channel activator BAY K 8644, suggesting that they lack L-type VOCCs. Rat and human TRH-Rs are highly conserved except at the C-terminus where the sequence differs. The C-terminus is believed to be important in receptor desensitization. Despite differences in this region, rat and human TRH-Rs expressed in HEK-293 cells underwent rapid (within 1 min) desensitization. This desensitization was dose-dependent and did not involve receptor loss. Similarly the bradykinin receptor endogenous to HEK-293 cells also displays a rapid desensitization. We conclude that in TRH-R-expressing non-pituitary HEK-293 cells, TRH mobilizes intracellular Ca2+ resulting in a monophasic Ca2+ transient. The rat and human TRH-Rs as well as the endogenous bradykinin receptor also displayed rapid receptor desensitization.
Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells
Authors: Patton W A , Granzow C A , Getts L A , Thomas S C , Zotter L M , Gunzel K A , Lowe-Krentz L J
Published: August 2015

The binding of heparin or heparan sulphate to a variety of cell types results in specific changes in cell function. Endothelial cells treated with heparin alter their synthesis of heparan sulphate proteoglycans and extracellular matrix proteins. In order to identify a putative endothelial cell heparin receptor that could be involved in heparin signalling, anti-(endothelial cell) monoclonal antibodies that significantly inhibit heparin binding to endothelial cells were prepared. Four of these antibodies were employed in affinity-chromatographic isolation of a heparin-binding protein from detergent-solubilized endothelial cells. The heparin-binding protein isolated from porcine aortic endothelial cells using four different monoclonal antibodies has an M(r) of 45,000 assessed by SDS/PAGE. The 45,000-M(r) heparin-binding polypeptide is isolated as a multimer. The antibody-isolated protein binds to heparin-affinity columns as does the pure 45,000-M(r) polypeptide, consistent with its identification as a putative endothelial heparin receptor.
Reconstitution and characterization of ATP-dependent bile acid transport in human and rat placenta
Authors: Bravo P , Marin J J G , Beveridge M J , Novak D A
Published: August 2015

Bile acid (BA) transport across the human microvillus maternal-facing trophoblast plasma membrane (mTPM) has been recently reported to be stimulated by the presence of ATP [Marin, Bravo, El-Mir and Serrano (1993) J. Hepatol. 18, S41]. Reconstitution of BA transport activity in proteoliposomes from human mTPM is reported in this paper. Typical characteristics of BA transport in native mTPM vesicles, including a requirement for ATP hydrolysis and inhibition by other BA species, were preserved in proteoliposome preparations. BA transport into 20- and 14-day-gestation rat mTPM vesicles was also stimulated by the presence of ATP as noted in human mTPM and in the rat liver canalicular membrane. Besides this functional similarity, these ATP-dependent carriers may share structural characteristics, as demonstrated by studies using an antibody (100 Ab) raised against the 100 kDa BA carrier of the canalicular membrane from rat liver which recognized proteins in both human and rat brush-border trophoblast membranes.
The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site
Authors: Murray J B , Adams C J , Arnold J R P , Stockley P G
Published: August 2015

We report details of the synthesis and characterization of oligoribonucleotides containing 4-thiouridine or 2-pyrimidinone ribonucleoside (4HC). We have used these probes to examine the roles of the conserved pyrimidines in the central core of the hammerhead ribozyme. The effects on catalysis of singly-substituted hammerhead ribozyme and substrate strands were quantified in multiple-turnover reactions. Various effects were observed on kcat. and Km, with up to a 7-fold decrease and a 3-fold increase respectively. For substitutions with 4HC at positions 3 or 17, catalytic activity in single turnover reactions can be increased up to 8-fold equivalent to 40% of wild-type activity, by increasing the concentration of the Mg2+ cofactor, implying that these substitutions had a deleterious effect on Mg2+ binding. Calculations of the change in the apparent free energy of binding for variants at positions 3, 4 or 17 are each consistent with deletion of a single hydrogen-bond to an uncharged group in the ribozyme. The cytidine 5′ to the scissile phosphate had not previously been thought to play a direct role in catalysis, however, removal of the exocyclic amino group decreased kcat. 4-fold. Recently, the crystal structures of a hammerhead ribozyme bound to either a non-cleavable 2′-deoxy substrate strand or a ribo-substrate strand have been reported. The kinetic properties of the variants described here are consistent with several key interactions seen in the crystals, in particular they provide experimental support for the assignment of the proposed catalytically active magnesium ion-binding site.
Agonists for endothelial P2 purinoceptors trigger a signalling pathway producing Ca2+ responses in lymphocytes adherent to endothelial cells
Authors: Wiley J S , Chen J R , Jamieson G P , Thurlow P J
Published: August 2015

Recirculation of lymphocytes through the body involves their frequent adhesion to endothelial cells but little is known of the signalling pathways between these two cell types. Lymphocytes from patients with chronic lymphocytic leukaemia were loaded with the Ca(2+)-sensitive indicator, fura 2, and allowed to adhere to either glass or monolayers of human umbilical-vein endothelial cells. Addition of ATP or UTP (1-10 microM) to the superfusate produced a transient rise in cytosolic Ca2+ concentration in the lymphocytes adherent to endothelium (24 of 35 cells). In contrast, ATP or UTP (1-10 microM) had no effect on the cytosolic Ca2+ of lymphocytes attached to glass. As the only lymphocyte receptor for ATP (P2Z class) requires higher ATP concentrations (> 50 microM) for Ca2+ influx and is unresponsive to UTP, the involvement of a lymphocyte P2Z purinoceptor is unlikely. Various agonists including ATP, UTP, 2-methylthioATP, ADP and histamine all stimulated increases in endothelial cytosolic Ca2+ but only ATP and UTP (both agonists for endothelial P2U purinoceptors) triggered Ca2+ transients in adherent lymphocytes. Removal of extracellular Ca2+ did not abolish the ATP-induced rise in cytosolic Ca2+ concentration in lymphocytes adherent to endothelial cells. These findings show that stimulation of endothelial P2U purinoceptors triggers an endothelial-lymphocyte signalling pathway which releases internal Ca2+ in adherent lymphocytes.
Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf
Authors: Bevilacqua M A , Faniello M C , D′Agostino P , Quaresima B , Tiano M T , Pignata S , Russo T , Cimino F , Costanzo F
Published: August 2015

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.
Two new sphingomyelin analogues inhibit phosphatidylcholine biosynthesis by decreasing membrane-bound CTP: phosphocholine cytidylyltransferase levels in HaCaT cells
Authors: Wieder T , Perlitz C , Wieprecht M , Huang R T C , Geilen C C , Orfanos C E
Published: August 2015

The effects of two newly synthesized sphingomyelin analogues on phosphatidylcholine biosynthesis were investigated in the immortalized human keratinocyte cell line HaCaT. N-Acetyl-erythro-sphingosine-1-phosphocholine (AcSM) and N-octanoyl-erythro-sphingosine-1-phosphocholine (OcSM) inhibited the incorporation of choline into phosphatidylcholine with half-inhibitory concentrations (IC50) of 6 micrograms/ml and 10 micrograms/ml respectively. Further experiments revealed that AcSM and OcSM interfered with the translocation of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), in HaCaT cells and inhibited cytidylyltransferase activity in vitro. Despite the fact that OcSM was a potent inhibitor of cytidylyltransferase in vitro, its effects on phosphatidylcholine biosynthesis and translocation of cytidylyltransferase in HaCaT cells were less pronounced as compared with AcSM. Finally, we showed that the comparatively strong effects of AcSM in cell culture experiments were due to the uptake of large amounts of this sphingomyelin analogue into the cells. The results presented demonstrate that the activity of cytidylyltransferase may be negatively regulated by a high ratio of choline head group-containing sphingolipids.
Abnormal type III collagen produced by an exon-17-skipping mutation of the COL3A1 gene in Ehlers-Danlos syndrome type IV is not incorporated into the extracellular matrix
Authors: Chiodo A A , Sillence D O , Cole W G , Bateman J F
Published: August 2015

A novel heterozygous mutation of the COL3A1 gene that encodes the alpha 1(III) chains of type III collagen was identified in a family with the acrogeric form of Ehlers-Danlos syndrome type IV (EDS-IV). Cultured dermal fibroblasts produced normal and shortened alpha 1(III) chains. The triple helix of the latter chain was shortened owing to a 33 amino acid deletion of Gly-184 to Pro-216. The corresponding region of cDNA lacked 99 base pairs from nucleotides 1051 to 1149. The deletions corresponded exactly to the normal sequence encoded by exon 17 of the COL3A1 gene. The proband was heterozygous for a T to G transversion at position +2 of intron 17, which resulted in skipping of exon 17. The splicing defect was not corrected by growing the fibroblasts at 33 degrees C and no other splicing variants were identified at 33 or 37 degrees C. The affected brother had the same mutation but his unaffected mother did not. Heterotrimeric type III collagen molecules containing normal and mutant chains were retained within the cell. The mutant homotrimeric molecules were modified and secreted normally and were thermally stable. These normal characteristics of the mutant homotrimers suggested that the loss of ten Gly-Xaa-Yaa triplets (where Gly-Xaa-Yaa is a repetitive amino acid triplet structure in which Xaa and Yaa are other amino acids, proline and hydroxyproline being more common in the Yaa position) did not adversely affect the formation and stability of the triple helix or the structural requirements for secretion. However, the mutant homotrimers were not incorporated into the extracellular matrix of an in vitro model of EDS-IV dermis. The EDS-IV phenotype in this family was probably due to a deficiency in the amount of normal type III collagen available for formation of the heterotypic collagen fibrils of the extracellular matrix. Intracellular and extracellular quality-control mechanisms prevented the incorporation of heterotrimeric and homotrimeric mutant type III collagen molecules into the cross-linked extracellular matrix.
Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum
Authors: Berger B , Hunziker P E , Hauer C R , Birchler N , Dallinger R
Published: August 2015

1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.
Factors affecting the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine
Authors: Milne J J , Malthouse J P G
Published: August 2015

13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20)-catalysed hydrogen-deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine. The first- and second-order rate constants for exchange when the alpha 2 beta 2 enzyme complex is or is not saturated with glycine have been determined at pH 7.0 and 7.8. At pH 7.8 the effects of binding the allosteric effector, DL-alpha-glycerol 3-phosphate, and of removing the alpha-subunits have been examined. The beta-subunits preferentially catalyse the exchange of the pro-2R proton of glycine, but adding alpha-subunits decreases the stereospecificity of the exchange reactions. Likewise, binding of DL-alpha-glycerol 3-phosphate to the alpha 2 beta 2 enzyme complex causes a further decrease in the stereospecificity of this reaction. The stereospecificity of the second-order exchange reaction catalysed by the beta-subunits is 136-fold larger than that of the alpha 2 beta 2 enzyme complex in the presence of DL-alpha-glycerol 3-phosphate, while there is only a 5-fold decrease in the stereospecificity of the first-order exchange reaction under the same conditions. We discuss how these results relate to current theories which attempt to explain how the alpha-subunits and DL-alpha-glycerol 3-phosphate modify the catalytic properties of tryptophan synthase.
Ionic regulation of endonuclease activity in PC12 cells
Authors: Villalba M , Ferrari D , Bozza A , Del Senno L , Di Virgilio F
Published: August 2015

We have investigated the Ca2+ dependency of DNA degradation into nucleosome-sized fragments in intact chromaffin-like PC12 cells and PC12 nuclear fractions. In intact cells we were unable to trigger DNA fragmentation by inducing either transient or sustained elevations of cytoplasmic Ca2+ ([Ca2+]i) with the Ca2+ ionophore ionomycin. On the contrary, DNA fragmentation was induced in intact cells by the intracellular Zn2+ chelator NNN'N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). To characterize further PC12 cell endonuclease activity, we then investigated digestion by purified PC12 cell fractions of exogenously added plasmids. In nuclear fractions two endonuclease activities were identified: an acidic (pH 5.0) endonuclease activity that was fully Ca2+- and Mg(2+)-independent; and a neutral (pH 7.6) endonuclease activity that was Ca(2+)-independent but Mg(2+)-dependent. Both endonuclease activities were inhibited by Zn2+. Nuclear membrane permeabilization greatly enhanced plasmid digestion at pH 7.6, but not at pH 5.0. This suggests that neutral endonuclease was located in a membrane-bound compartment, whereas acidic endonuclease was freely accessible to the substrate even in the presence of an intact nuclear membrane. In intact nuclei, digestion of genomic DNA could not be triggered by increasing the bivalent cation composition of the medium. On the contrary, in hypotonic medium we observed a large spontaneous nucleolytic DNA degradation that was increased by Zn2+ chelation. However, an acidic pH shift was a potent stimulus for DNA fragmentation in isotonic as well as hypotonic medium.
Metabolism of [3-13C]pyruvate and [3-13C]propionate in normal and ischaemic rat heart in vivo: 1H- and 13C-NMR studies
Authors: Sumegi B , Podanyi B , Forgo P , Kover K E
Published: August 2015

The oxidation of [3-13C]pyruvate and [3-13C]propionate was studied in vivo in infused rats. The infused [3-13C]pyruvate was quickly converted to [3-13C]lactate in the blood, and the [3-13C]lactate formed was well metabolized in both normoxic and ischaemic hearts. Large differences (200-600%) in the 13C enrichment of alanine (C-3) and acetyl-CoA (C-2) compared with lactate (C-3) were found in both normoxic and ischaemic hearts, suggesting that the extracellular [3-13C]lactate preferentially entered a region of the cytoplasm which specifically transfers the labelled pyruvate (formed from [3-13C]lactate) to the mitochondria. The highly enriched mitochondrial pyruvate gave high enrichment in alanine and acetyl-CoA, which was detected by 1H- and 13C-NMR spectroscopy. Ischaemia increased 13C incorporation into the main cytoplasmic lactate pool and decreased 13C incorporation into citric acid cycle intermediates, mainly decreasing the pyruvate anaplerosis. Isoprenaline-induced ischaemia of the heart caused only a slight decrease in pyruvate oxidation. In contrast to the decreased anaplerosis of pyruvate, the anaplerosis of propionate (and propionyl-carnitine) increased significantly in ischaemic hearts, which may contribute to the protective effect of propionyl-carnitine seen in ischaemia. In addition, we found that [3-13C]propionate preferentially labelled aspartate C-3 in rat heart, suggesting incomplete randomization of label in the succinyl-CoA-malate span of the citric acid cycle. These data show that proton observed 13C edited spectroscopic methods, i.e. heteronuclear spin-echo and the one-dimensional heteronuclear multiple quantum coherence sequence, can be successfully used to study heart metabolism in vivo.
A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus
Authors: Schwartz B , Hittelman A , Daneshvar L , Basu H S , Marton L J , Feuerstein B G
Published: August 2015

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.