bluuebunny/crossref_metadata_2025 huggingface dataset:
The plasma membrane of Leishmania donovani promastigotes is the main target for CA(1–8)M(1–18), a synthetic cecropin A–melittin hybrid peptide
Authors: DÍAZ-ACHIRICA Pilar , UBACH Josep , GUINEA Almudena , ANDREU David , RIVAS Luis
Published: August 2015

Reports on the lethal activity of animal antibiotic peptides have largely focused on bacterial rather than eukaryotic targets. In these, involvement of internal organelles as well as mechanisms different from those of prokaryotic cells have been described. CA(1-8)M(1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. Using Leishmania donovani promastigotes as a model system we have studied the mechanism of action of CA(1-8)M(1-18), its two parental peptides and two analogues. At micromolar concentration CA(1-8)M(1-18) induces a fast permeability to H+/OH-, collapse of membrane potential and morphological damage to the plasma membrane. Effects on other organelles are related to the loss of internal homeostasis of the parasite rather than to a direct effect of the peptide. Despite the fast kinetics of the process, the parasite is able to deactivate in part the effect of the peptide, as shown by the higher activity of the d-enantiomer of CA(1-8)M(1-18). Electrostatic interaction between the peptide and the promastigote membrane, the first event in the lethal sequence, is inhibited by polyanionic polysaccharides, including its own lipophosphoglycan. Thus, in common with bacteria, the action of CA(1-8)M(1-18) on Leishmania promastigotes has the same plasma membrane as target, but is unique in that different peptides show patterns of activity that resemble those observed on eukaryotic cells.
Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades
Authors: GARNOVSKAYA N. Maria , MUKHIN Yurii , RAYMOND R. John
Published: August 2015

These experiments tested the hypothesis that signalling elements involved in the activation of the extracellular signal-regulated protein kinase (ERK) mediate rapid activation of sodium-proton exchange (NHE) in fibroblasts when both signals are initiated by a single G protein-coupled receptor, the 5-HT1A receptor. Similarities between the two processes were comparable concentration-response curves and time-courses, and overlapping sensitivity to some pharmacological inhibitors of tyrosine kinases (staurosporine and genistein), and phosphoinositide 3ʹ-kinase (wortmannin and LY204002). Activation of NHE was much more sensitive to the phosphatidylcholine-specific phospholipase inhibitor (D609) than was ERK. Neither pathway was sensitive to manoeuvres designed to block PKC. In contrast, Src or related kinases appear to be required to activate ERK, but not NHE. Transfection of cDNA constructs encoding inactive mutant phosphoinositide 3ʹ-kinase, Grb2, Sos, Ras, and Raf molecules were successful in attenuating ERK, but had essentially no effect upon NHE activation. Finally, PD98059, an inhibitor of mitogen activated/extracellular signal regulated kinase kinase, blocked ERK but not NHE activation. Thus, in CHO fibroblast cells, activation by the 5-HT1A receptor of ERK and NHE share a number of overlapping features. However, our studies do not support a major role for ERK, when activated by the 5-HT1A receptor, as a short-term upstream regulator of NHE activity.
Estimation of total rate of formation of nitric oxide in the rat
Authors: SAKINIS Augustinas , WENNMALM Åke
Published: August 2015

Nitric oxide (NO) is a powerful mediator with important actions in several organ systems. NO is synthesized during the enzymatic conversion of L-arginine and molecular oxygen to l-citrulline. About 90% of the NO formed is degraded to nitrate. Utilizing this information we have developed a method for assessment of the total rate of formation of NO in the rat. Male Wistar rats were kept in a closed-cage system allowing controlled breathing of a mixture of 18O2 and 16O2 in N2 for up to 5 h. Blood samples for mass spectrometric analysis of nitrate residues with varying numbers of 18O atoms incorporated were drawn before and during the exposure to 18O2. By comparing the relative incorporation of 18O into nitrate residues to the 16O2/18O2 ratio in the breathing gas mixture in the cage system it was possible to calculate the absolute rate of NO formation in the animal. The rate of formation of NO in anaesthetized rats ranged from 0.33 to 0.85 μmol·kg-1·h-1. The rate of formation did not differ significantly in rats which were awake during the experiment (range 0.36-0.72 μmol·kg-1·h-1). The L-arginine analogue Nω-nitro-L-arginine methyl ester (L-NAME) dose-dependently inhibited the formation of NO, at a dose of 100 mg/kg by more than 99%. The technique presented allows estimation of the total rate of formation of NO in vivo in rats. Application of the technique may yield important information about the physiological and pathophysiological roles of NO. It may also be utilized to evaluate the effect of pharmacological treatment on NO formation.
Identification of a Caenorhabditis elegans Δ6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae
Authors: NAPIER A. Johnathan , HEY J. Sandra , LACEY J. Dominic , SHEWRY R. Peter
Published: August 2015

We identified a cDNA expressed sequence tag from an animal (the nematode worm Caenorhabditis elegans) that showed weak similarity to a higher-plant microsomal Δ6-desaturase. A full-length cDNA clone was isolated and expressed in the yeast Saccharomyces cerevisiae. This demonstrated that the protein encoded by the C. elegans cDNA was that of a fatty acid Δ6-desaturase, as determined by the accumulation of γ-linolenic acid. The C. elegans Δ6-desaturase contained an N-terminalcytochrome b5 domain, indicating that it had a similar structure to that of the higher-plant Δ6-desaturase. The C. elegans Δ6-desaturase mapped to cosmid W08D2, a region of chromosome III. This is the first example of a Δ6-desaturase isolated from an animal and also the first example of an animal desaturase containing a cytochrome b5 domain.
Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells
Authors: MIRALEM Tiho , TEMPLETON M. Douglas
Published: August 2015

Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partially suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbol ester or platelet-derived growth factor, this response appears to result from the ability of heparin to suppress activation of the extracellular-signal-regulated kinase family of mitogen-activated protein kinases. However, we have also shown that heparin suppresses c-fos expression in response to ionophores such as ionomycin, an event independent of mitogen-activated protein kinase [Miralem, Wang, Whiteside and Templeton (1996) J. Biol. Chem.271, 17100-17106]. Here we identify this second heparin-sensitive pathway as involving Ca2+/calmodulin-dependent kinase (CaMK) II. Ionomycin (100 nM) caused a transient rise in intracellular Ca2+ concentration ([Ca2+]i) in quiescent RMCs to 386±55 nM, with an increase in CaMK II activity that peaked 30 s later. The accumulation of c-fos mRNA that ensued 30 min later was prevented when the increase in [Ca2+]i was prevented with the intracellular Ca2+ chelator, 1,2-bis-(2-aminophenyoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic acid. The broad-specificity CaMK inhibitor, KT 5926, inhibited ionomycin-dependent c-fos induction at a concentration at which it was without effect on induction by serum or phorbol ester. The CaMK II-specific inhibitor, KN-93, likewise inhibited c-fos induction by ionomycin, but not by serum or phorbol ester. ML-7, an inhibitor of the CaMK-related myosin light-chain kinase (MLCK), was without effect. Heparin (1 μg/ml) suppressed ionomycin-dependent c-fos induction. It was without effect on [Ca2+]i, but inhibited the development of autonomous CaMK II activity. However, when heparin was added to the CaMK II assay solution in vitro, it was without effect on autonomous activity. Furthermore, heparin did not prevent full activation of CaMK II by the Ca2+-calmodulin complex in vitro. Heparin did not affect myosin light-chain phosphorylation or RMC contraction, processes mediated by MLCK. We conclude that ionomycin induces c-fos in RMCs through the CaMK II pathway, and that heparin prevents CaMK II activation by an indirect process mediated by other cell components. Heparin does not affect activation of the closely related CaMK, MLCK.
Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes
Authors: SPRANGERS Fleur , SAUERWEIN P. Hans , ROMIJN A. Johannes , van WOERKOM M. George , MEIJER J. Alfred
Published: August 2015

There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at other levels. Using hepatocytes from fasted rats incubated with the NO-donor S-nitroso-N-acetylpenicillamine, we have now found that the synthesis of glycogen from glucose is even more sensitive to inhibition by NO than gluconeogenesis. Inhibition of glycogen production by NO was accompanied by a rise in intracellular glucose 6-phosphate and UDPglucose. Activity of glycogen synthase, as measured in extracts of hepatocytes after the cells had been exposed to NO, was decreased. Experiments with gel-filtered liver extracts revealed that inhibition of glycogen synthase was caused by an inhibitory effect of NO on the conversion of glycogen synthase b into glycogen synthase a.
Identification of the major allergens in wheat flour responsible for baker's asthma
Authors: AMANO Maho , OGAWA Haruko , KOJIMA Kyoko , KAMIDAIRA Tomoko , SUETSUGU Susumu , YOSHIHAMA Munehiro , SATOH Takanori , SAMEJIMA Tatsuya , MATSUMOTO Isamu
Published: August 2015

Baker's asthma, a typical occupational allergic disease, is a serious problem in the food industries. In this study, purification and identification of major allergens recognized by IgEs in sera of allergic patients were performed. Major immunoreactive proteins were purified from the albumin fraction by gel filtration on a Toyopearl HW-50 column followed by reverse-phase HPLC. The N-terminal amino acid sequences and molecular masses measured by MS indicated that the major immunoreactive proteins are members of the α-amylase inhibitor family, 0.19 and 0.28. Significant leukotriene release by each purified protein was observed in cell-associated stimulation tests, suggesting in vivo activity of these antigens. Carbohydrate analyses of major allergens indicated that they are monoglycosylated but not N-glycosylated in spite of the presence of a potential N-glycosylation site. Recombinant 0.19 expressed in Escherichia coli showed the same reactivity with IgE as native wheat 0.19 in Western blotting and ELISA using methyl vinyl ether maleic anhydride co-polymer as an immobilizing reagent, suggesting that the allergenic epitopes are located in the peptide portions.
Mechanism of reaction of myoglobin with the lipid hydroperoxide hydroperoxyoctadecadienoic acid
Authors: REEDER J. Brandon , WILSON T. Michael
Published: August 2015

The reaction between myoglobin and the lipid hydroperoxide 13(S)-hydroperoxy-9,11(cis,trans)-octadecadienoic acid (HPODE) was studied kinetically by spectrophotometric, polarographic and analytical methods. Metmyoglobin catalysed the decomposition of HPODE, resulting in peroxide, oxygen and conjugated diene depletion, together with the transient production of ferryl myoglobin. The reaction stoichiometry was 2:1:1 for peroxide to oxygen to conjugated diene, whereas the myoglobin remained generally intact. This stoichiometry and the rates of change of conjugated diene and ferryl myoglobin concentrations were not completely consistent with previously proposed mechanisms. We propose a novel mechanism in which HPODE reacts with both ferric myoglobin and ferryl myoglobin to form a redox cycle. Both peroxyl and alkoxyl radicals are produced, explaining the observed stoichiometry of peroxide, oxygen and conjugated diene depletion and the transient appearance of ferryl myoglobin. Computer simulation shows that this mechanism is fully capable of reproducing the observed time courses of all components.
Human CD38 is an authentic NAD(P)+ glycohydrolase
Authors: BERTHELIER Valérie , TIXIER Jean-Michel , MULLER-STEFFNER Hélène , SCHUBER Francis , DETERRE Philippe
Published: August 2015

The leucoyte surface antigen CD38 has been shown to be an ecto-enzyme with multiple catalytic activities. It is principally a NAD+ glycohydrolase that transforms NAD+ into ADP-ribose and nicotinamide. CD38 is also able to produce small amounts of cyclic ADP-ribose (ADP-ribosyl cyclase activity) and to hydrolyse this cyclic metabolite into ADP-ribose (cyclic ADP-ribose hydrolase activity). To classify CD38 among the enzymes that transfer the ADP-ribosyl moiety of NAD+ to a variety of acceptors, we have investigated its substrate specificity and some characteristics of its kinetic and molecular mechanisms. We find that CD38-catalysed cleavage of the nicotinamide-ribose bond results in the formation of an E·ADP-ribosyl intermediary complex, which is common to all reaction pathways; this intermediate reacts (1) with acceptors such as water (hydrolysis), methanol (methanolysis) or pyridine (transglycosidation), and (2) intramolecularly, yielding cyclic ADP-ribose with a low efficiency. This reaction scheme is also followed when using nicotinamide guanine dinucleotide as an alternative substrate; in this case, however, the cyclization process is highly favoured. The results obtained here are not compatible with the prevailing model for the mode of action of CD38, according to which this enzyme produces first cyclic ADP-ribose which is then immediately hydrolysed into ADP-ribose (i.e. sequential ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities). We show instead that the cyclic metabolite was a reaction product of CD38 rather than an obligatory reaction intermediate during the glycohydrolase activity. Altogether our results lead to the conclusion that CD38 is an authentic ‘classical’ NAD(P)+ glycohydrolase (EC 3.2.2.6).
A phogrin–aequorin chimaera to image free Ca2+ in the vicinity of secretory granules
Authors: POULI E. Aristea , KARAGENC Nedim , WASMEIER Christina , HUTTON C. John , BRIGHT Nick , ARDEN Sue , SCHOFIELD J. George , RUTTER A. Guy
Published: August 2015

Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to the control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2+] in the outer mantle (< 30 nm deep) that surrounds the surface of dense-core secretory granules, we have designed a recombinant chimaera between the granule protein phogrin and aequorin. When expressed in populations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombinant protein reported a similar [Ca2+] at the granule surface to that in the bulk cytosol, measured with untargeted aequorin. This was the case both at rest ([Ca2+] = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools, and during activated secretion. Thus depolarization of MIN6 cell populations with high K+ increased [Ca2+] both in the bulk cytosol and close to the granules to approx. 4 μM, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in [Ca2+] in either domain to 1.3 μM. These data argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mobilization of Ca2+ from most secretory granules probably does not occur during activated Ca2+ influx or mobilization of internal Ca2+ stores, and (ii) agonist-stimulated Ca2+-dependent secretion can occur without development of a large gradient of [Ca2+] between the surface of most secretory vesicles and the rest of the cytosol.
Isolation of a novel bone glycosylated phosphoprotein with disulphide cross-links to osteonectin
Authors: ZHOU Hai-Yan , SALIH Erdjan , GLIMCHER J. Melvin
Published: August 2015

An 80 kDa protein was purified from calf bone by HCl-demineralization followed by 0.5 M EDTA/1.0 M NaCl extraction and sequential chromatography on DE-52, hydroxyapatite, and TSK-gel G3000SW HPLC columns. From the DE-52 column the protein was eluted at three different fractions, of which one further separated into two fractions on the hydroxyapatite column, indicating that the protein is present in four different molecular forms designated as 80 k-I-1, k-I-2, k-II, k-III. The N-terminal sequence analysis of all four forms gave the same sequence, SEQYNQEPNNV. Several tryptic internal peptides were also generated, purified and sequenced, leading to the identification of several repeat sequences, IFLGXXEI. Homology searching of the N-terminal and internal sequences indicates that this is a novel protein. Both 80 k-I-2 and k-III had similar amino acid composition with high contents of Asx, Glx and Leu and contained 7 and 16 phosphoserines per 1000 total amino acids, respectively. The 80 k-I-1 and 80 k-II forms were stained with Rhodamine B specific for phosphoproteins. The four forms contained different contents of neutral sugars ranging from 5.5 to 26% (w/w protein) and ~ 1.7% sialic acid. These data indicated that the 80 kDa protein exists in four isomeric forms, at least based on the different post-translational modifications. The evaluation of the 80 kDa glycosylated phosphoprotein under alkylating, reducing and non-reducing conditions indicated that this protein undergoes polymerization through intermolecular disulphide bonds. Furthermore, the 80 kDa protein and osteonectin (ON), both of which are cysteine-rich proteins, can cross-link with each other via disulphide bonds, and this process can be induced to take place in vitro under experimental conditions. The occurrence of such a phenomenon in vivo was confirmed from the presence of similar high Mr components containing both 80 kDa and ON in the same SDS/PAGE bands, detected by the respective antibody reactions in crude bone extracts which were extracted in the presence of alkylating agent.
Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes
Authors: OGBOURNE Steven , ANTALIS Toni M.
Published: August 2015

Mechanisms controlling transcription and its regulation are fundamental to our understanding of molecular biology and, ultimately, cellular biology. Our knowledge of transcription initiation and integral factors such as RNA polymerase is considerable, and more recently our understanding of the involvement of enhancers and complexes such as holoenzyme and mediator has increased dramatically. However, an understanding of transcriptional repression is also essential for a complete understanding of promoter structure and the regulation of gene expression. Transcriptional repression in eukaryotes is achieved through ‘silencers ’, of which there are two types, namely ‘silencer elements ’ and ‘negative regulatory elements ’ (NREs). Silencer elements are classical, position-independent elements that direct an active repression mechanism, and NREs are position-dependent elements that direct a passive repression mechanism. In addition, ‘repressors ’ are DNA-binding trasncription factors that interact directly with silencers. A review of the recent literature reveals that it is the silencer itself and its context within a given promoter, rather than the interacting repressor, that determines the mechanism of repression. Silencers form an intrinsic part of many eukaryotic promoters and, consequently, knowledge of their interactive role with enchancers and other transcriptional elements is essential for our understanding of gene regulation in eukaryotes.
Dietary carbohydrates enhance lactase/phlorizin hydrolase gene expression at a transcription level in rat jejunum
Authors: TANAKA Takemi , KISHI Kazuhiro , IGAWA Makiko , TAKASE Sachiko , GODA Toshinao
Published: August 2015

We have previously shown that dietary sucrose stimulates the lactase/phlorizin hydrolase (LPH) mRNA accumulation along with a rise in lactase activity in rat jejunum [Goda, Yasutake, Suzuki, Takase and Koldovský (1995) Am. J. Physiol. 268, G1066–G1073]. To elucidate the mechanisms whereby dietary carbohydrates enhance the LPH mRNA expression, 7-week-old rats that had been fed a low-carbohydrate diet (5.5% of energy as starch) were given diets containing various monosaccharides or sucrose for 12 h. Among carbohydrates examined, fructose, sucrose, galactose and glycerol elicited an increase in LPH mRNA accumulation along with a rise in lactase activity in the jejunum. By contrast, glucose and α-methylglucoside were unable to elicit a significant increase in LPH mRNA levels. To explore a transcriptional mechanism for the carbohydrate-induced increases in LPH mRNA levels, we employed two techniques currently available to estimate transcriptional rate, i.e. RNA protection assays of pre-mRNA using an intron probe, and nuclear run-on assays. Both assays revealed that fructose elicited an increase in transcription of the LPH gene, and that the transcription of LPH was influenced only slightly, if at all, by glucose intake. These results suggest that certain monosaccharides such as fructose or their metabolite(s) are capable of enhancing LPH mRNA levels in the small intestine, and that transcriptional control might play a major role in the carbohydrate-induced increase of LPH mRNA expression.
Metabolism and apoptotic properties of elevated ceramide in HT29rev cells
Authors: VELDMAN Robert J. , KLAPPE Karin , HOEKSTRA Dick , KOK Jan W.
Published: August 2015

Ceramide (Cer) has been implicated in the regulation of apoptosis. In this study, we elevated cellular Cer levels in human colon-carcinoma (HT29rev) cells by incubating the cells in the presence of bacterial sphingomyelinase (bSMase) or, alternatively, in the presence of C2-Cer, a short-chain analogue of the sphingolipid. bSMase treatment did not induce apoptosis in these cells, as revealed by a lack of both DNA fragmentation and cleavage of poly(ADP-ribose)polymerase. In contrast, apoptosis did occur upon addition of C2-Cer. These findings led us to study whether differences in the metabolic fate of the excess of Cer, as generated by both treatments, contributed to the observed difference in apoptosis-inducing capacity. C2-Cer was rapidly taken up by HT29rev cells and accumulated due to the absence of substantial metabolic conversion. Upon addition of bSMase, hydrolysis of sphingomyelin resulted in a reduction of that pool to 20% compared with control values, accompanied by a multi-fold increase in Cer level. In spite of the continuous presence of active bSMase, the Cer increase turned out to be transient. Cer levels reached their maximum 1–2 h after addition of bSMase, followed by a significant decrease. Excessive Cer was mainly turned over via cerebrosides into complex glycolipids, including gangliosides. In the presence of glucosylceramide synthase- and/or ceramidase inhibitors, this conversion was significantly blocked and bSMase-generated Cer accumulated in the cells. However, even under these conditions apoptosis did not occur. In conclusion, the inability of bSMase to induce apoptosis of HT29rev cells does not appear to be due to rapid metabolic conversion of excessive Cer. Since apoptosis is induced upon addition of C2-Cer, we therefore propose that the intracellular target involved in the propagation of the apoptotic signal is reached by C2-Cer, but not by bSMase-generated Cer.
Mucus glycoproteins from pig gastric mucosa: different mucins are produced by the surface epithelium and the glands
Authors: NORDMAN Henrik , DAVIES Julia R. , CARLSTEDT Ingemar
Published: August 2015

An antibody (PGM2B) recognizing a pig gastric-mucin apoprotein reacts with the surface epithelium of pig gastric mucosa. Virtually no reactivity was observed over the mucin-producing cells in the glands, which were recognized by the GlcNAc-selective Griffonia simplicifoliaII (GSA-II) lectin. Mucins from the glandular tissue of the cardiac region, corpus and antrum were purified using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. In the cardiac region, two major mucin populations at 1.5 and 1.4 g/ml were identified. The high-density population reacted preferentially with the PGM2B antibody and resembled mucins from the surface epithelium of this region, whereas the low-density population reacted strongly with the GSA-II lectin and appeared to originate from the glands. In the glandular tissue of corpus, a component with strong GSA-II lectin reactivity, which was distinctly different from the surface mucins from this region, was found at 1.4 g/ml, thus resembling the gland component from the cardiac region. Mucins from antrum glandular tissue contained at least two GSA-II lectin-reactive populations banding at 1.5 and 1.4 g/ml, respectively. Gland mucins from all regions were large oligomeric glycoproteins and heterogeneous with respect to charge properties, as shown by using rate-zonal centrifugation and ion-exchange HPLC, respectively. Gel chromatography of mucin glycopeptides showed that gland mucins from antrum and corpus contained significantly longer glycosylated domains than those from the surface mucosa. Thus, mucins from pig gastric glandular tissue comprise a number of large and oligomeric glycoproteins that differ from those from the surface epithelium in buoyant density, apoprotein structure and carbohydrate substitution.
LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function
Authors: SCHMEICHEL Karen L. , BECKERLE Mary C.
Published: August 2015

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16–23HX2CX2CX2CX16–23CX2–3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein–protein interactions. For example, in the case of the zyxin–CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.
Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology
Authors: LACABARATZ-PORRET Christine , CORVAZIER Elisabeth , KOVÀCS Tünde , BOBE Régis , BREDOUX Raymonde , LAUNAY Sophie , PAPP Béla , ENOUF Jocelyne
Published: August 2015

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein–protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.
A novel transforming growth factor β2 antisense transcript in mammalian lung
Authors: COKER Robina K. , LAURENT Geoffrey J. , DABBAGH Karim , DAWSON James , McANULTY Robin J.
Published: August 2015

Transforming growth factor (TGF) β2 gene expression was examined in murine, rat and human lung by in situ hybridization with riboprobes. Hybridization signal was observed in a variety of cells with the sense probe, and Northern-blot analysis with this probe demonstrated the presence of a novel 3.5 kb transcript. This first report suggesting the existance of a natural TGFβ2 antisense transcript raises the possibility that such a transcript may play a role in regulating TGFβ2 production.
cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: generation of a molecular model
Authors: HOFFMANN Ralf , WILKINSON Ian R. , McCALLUM J. Fraser , ENGELS Peter , HOUSLAY Miles D.
Published: August 2015

Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram {4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone} inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54 → Asp mutant, with an IC50 value that was ∼ 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54 → Thr, Ser-54 → Tyr and Ser-54 → Cys mutants. The Glu-53 → Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54 → Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53 → Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51 → Ala/Arg-52 → Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54 → Asp and Ser-54 → Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion-pair interaction involving the negatively charged Glu-53. The increase in susceptibility to inhibition by rolipram upon PKA-mediated phosphorylation is suggested to involve the disruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.
A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity
Authors: DENNIS Caitríona A. , VIDELER Hortense , PAUPTIT Richard A. , WALLIS Russell , JAMES Richard , MOORE Geoffrey R. , KLEANTHOUS Colin
Published: August 2015

We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 Å resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.
Acute desensitization of phospholipase C-coupled muscarinic M3 receptors but not gonadotropin-releasing hormone receptors co-expressed in αT3-1 cells: implications for mechanisms of rapid desensitization
Authors: WILLARS Gary B. , McARDLE Craig A. , NAHORSKI Stefan R.
Published: August 2015

In the present study we have expressed the muscarinic M3 receptor in an immortalized mouse pituitary cell line (αT3-1), which expresses an endogenous gonadotropin-releasing hormone (GnRH) receptor, to examine potential differences in acute receptor regulation. Both of these receptors couple to the activation of phosphoinositide-specific phospholipase C (PLC) in these cells and we demonstrate that, despite expression in the same cell background, acute desensitization is a feature of muscarinic M3 receptors but not of GnRH receptors. We show that, when the concentrations of GnRH and methacholine are matched to give approximately equivalent maximal elevations of Ins(1,4,5)P3, the GnRH receptor is able to sustain PLC activity at the initial rate, whereas the muscarinic M3 receptor cannot. Thus PLC-activating G-protein-coupled receptors are able to undergo rapid desensitization in this cell line, indicating that the desensitization profile is receptor-specific rather than cell-specific. This argues strongly that post-receptor regulatory features do not have a prominent role in mediating rapid desensitization in these cells. Furthermore GnRH receptor-mediated PLC activity is sustained despite a marked and persistent depletion in the steady-state level of PtdIns(4,5)P2. In contrast, activation of muscarinic receptors is not sustained despite only a transient decrease in PtdIns(4,5)P2 concentration. Thus, whereas the contribution of PtdIns(4,5)P2 depletion to the temporal profile of receptor-mediated PLC signalling has been difficult to assess, the present results demonstrate that this is unlikely to be of importance in these cells. We suggest that unique structural features of the GnRH receptor result in a lack of appropriate regulatory phospho-acceptor sites and that the absence of agonist-dependent phosphorylation might underlie the lack of acute regulation.
Active Lyn protein tyrosine kinase is selectively enriched within membrane microdomains of resting platelets
Authors: DORAHY Douglas J. , BURNS Gordon F.
Published: August 2015

Circulating platelets are primed to respond very rapidly to thrombogenic stimuli, but most platelets complete their lifespan without ever becoming activated. Platelet activation is accompanied by waves of sequential tyrosine phosphorylation thought to involve members of the Src family of protein tyrosine kinases (PTKs). We show here that resting platelets contain highly active pp53/56Lyn PTK within membrane microdomains (rafts) isolated biochemically with or without the use of detergent. This fraction is also greatly enriched in the transmembrane glycoprotein CD36, known to associate with Lyn PTK, but in transfection studies we could find no evidence to suggest that CD36 affects the distribution or function of Lyn. Upon platelet activation Lyn activity remains constant or diminishes and pp60c-src PTK within this fraction becomes highly activated, indicating the dynamic nature of the membrane microdomains. It is suggested that the function of active Lyn PTK in the resting platelet is to allow prolonged survival of this anucleate cell.
T-cell-receptor signalling in inositol 1,4,5-trisphosphate receptor (IP3R) type-1-deficient mice: is IP3R type 1 essential for T-cell-receptor signalling?
Authors: HIROTA Junji , BABA Masashi , MATSUMOTO Mineo , FURUICHI Teiichi , TAKATSU Kiyoshi , MIKOSHIBA Katsuhiko
Published: August 2015

Stimulation of T-cells via the T-cell receptor (TCR) complex is accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i). Recently, it was reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of inositol 1,4,5-trisphosphate receptor (IP3R) type 1 (IP3R1), failed to demonstrate increased [Ca2+]i or interleukin-2 production after TCR stimulation and was also resistant to apoptotic stimuli. This cell line lacked IP3R1 expression, but expressed the type-2 and -3 receptors, IP3R2 and IP3R3 respectively [Jayaraman, Ondriasova, Ondrias, Harnick and Marks (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6007–6011, and Jayaraman and Marks (1997) Mol. Cell. Biol. 17, 3005–3012]. The authors concluded that IP3R1 is essential for TCR signalling and suggested that Ca2+ release via IP3R1 is a critical mediator of apoptosis. To establish whether a loss of IP3R1 function in T-cells occurred in vivo and in vitro, we investigated Ca2+ signalling after TCR stimulation and the properties of T-cells using IP3R1-deficient (IP3R1-/-) mice. As IP3R1-/- mice die at weaning, we transplanted bone marrow cells of IP3R1-/- mice into irradiated wild-type mice. Western blot analysis showed that the recipient IP3R1-containing (IP3R1+/+) lymphocytes were replaced by the donor IP3R1-/- lymphocytes after transplantation and that expression of IP3R2 and IP3R3 was unaltered. In contrast with the previous reports, T-cells lacking IP3R1 were able to mobilize Ca2+ from intracellular Ca2+ stores after stimulation via the TCR. We observed no significant differences between IP3R1+/+ and IP3R1-/- T-cells in terms of the number of thymocytes and splenocytes, the proportion of the T-cell phenotype, proliferative response to anti-CD3 monoclonal antibody (mAb) stimulation and cell viability. Therefore IP3R1 is not essential for T-cell development and function.
Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2
Authors: SULLIVAN Michael , RENA Graham , BEGG Fiona , GORDON Laurie , OLSEN Anne S. , HOUSLAY Miles D.
Published: August 2015

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5´–3´, telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active ‘long ’ and ‘short ’ isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5´ exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5´ exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 µM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 µM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitrotranscription–translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.
The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism
Authors: PÁRRAGA Antonio , GARCÍA-SÁEZ Isabel , WALSH Sinead B. , MANTLE Timothy J. , COLL Miquel
Published: August 2015

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.
Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes
Authors: MODAN-MOSES Dalit , JANICOT Michel , McLENITHAN John C. , LANE M. Daniel , CASELLA Samuel J.
Published: August 2015

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50–70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (α2,β2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor β subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.
Membrane composition can influence the rate of Al3+-mediated lipid oxidation: effect of galactolipids
Authors: VERSTRAETEN Sandra V. , KEEN Carl L. , GOLUB Mari S. , OTEIZA Patricia I.
Published: August 2015

In the first part of the present study we investigated the effects of pre-natal and early postnatal exposure of mice to high levels of dietary Al3+ on myelin lipid composition and lipid oxidation. We found: (1) a significantly higher (104%; P< 0.01) content of brain myelin galactolipids in the high-Al3+ group than in controls, and, (2) a significant correlation (r2 = 0.70; P< 0.01) between the concentration of myelin galactolipids and TBARS (2-thiobarbituric acid-reactive substances) content, a parameter of lipid oxidation. Based on these results, we evaluated in an in vitro model (liposomes) whether galactolipids could affect the capacity of Al3+ to stimulate Fe2+-initiated lipid oxidation, and whether this effect could be due to the promotion of changes in membrane physical properties (membrane phase separation and rigidification). The presence of galactolipids (10–40 mol%) in the liposomes caused a concentration-dependent increase in the stimulatory effect of Al3+ on Fe2+-induced TBARS production, and on the ability of Al3+ to induce phase separation and membrane rigidification. The capacity of Al3+ (10–100 µM) to induce lateral phase separation in liposomes composed of phosphatidylcholine/phosphatidylserine/galactolipid (36:24:40, molar ratio) was correlated significantly (r2 = 0.99; P< 0.001) with the stimulatory action of Al3+ on Fe2+-induced TBARS production. We propose that the high content of galactolipids found in myelin from Al3+-intoxicated mice could favour Al3+-induced changes in membrane physical properties, with the subsequent acceleration of lipid oxidation rates.