bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of Gαq and Gα11 gene expression in platelets and erythrocytes. Expressions of cellular differentiation and species differences
Authors: JOHNSON Gerhard J. , LEIS Linda A. , DUNLOP Patricia C.
Published: August 2015

Gαq and Gα11, members of the Gq family of G-proteins, transduce signals from receptors to the β isoenzymes of phosphatidylinositol-specific phospholipase C (PI-PLC). The receptor specificity of these α subunits is unknown. Gαq and Gα11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of Gα11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via Gαq, but the role of Gα11 is uncertain. To define their roles in platelet activation we studied Gαq and Gα11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT–PCR) and direct sequencing. An antiserum specific for mouse Gα11 failed to identify Gα11 in dog or human platelets or in dog liver, a tissue known to contain Gα11. RT–PCR performed with gene-specific primers demonstrated Gαq mRNA, but not Gα11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize Gα11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate Gα11, but not Gαq, mRNA. Compared with mouse cDNA, dog and human Gα11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as Gαq. Gαq mRNA was also found in mature erythrocytes but Gα11 mRNA was not identified, whereas both Gαq and Gα11 mRNAs were found in bone marrow stem cells. Therefore Gα11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of Gα11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via Gαq, and that Gα11 deficiency is not responsible for defective activation of PI-PLC in thromboxane-insensitive dog platelets. Despite the high degree of similarity that exists between Gαq and Gα11, significantly greater species-specific variation in nucleotide sequence is present in Gα11 than in Gαq. Cellular specificity and species specificity are important characteristics of these Gq family G-proteins.
Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum
Authors: WITTER Klaus , CAHILL Dolores J , WERNER Thomas , ZIEGLER Irmgard , RÖDL Wolfgang , BACHER Adelbert , GÜTLICH Markus
Published: August 2015

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 °C. The Km value for GTP is 50 µM.
Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells
Authors: KUGE Osamu , SAITO Kyoko , KOJIMA Michiyuki , AKAMATSU Yuzuru , NISHIJIMA Masahiro
Published: August 2015

We have isolated a full-length cDNA clone of the Chinese hamster ovary (CHO) pssC gene, which encodes mitochondrial phosphatidylserine decarboxylase. The cDNA clone is capable of increasing phosphatidylserine decarboxylase activity to 11-fold in CHO-K1 cells. The pssC gene product predicted from the cDNA sequence is composed of 409 amino acid residues. In an in vitro translation system coupled with in vitro transcription, the cDNA clone directs the formation of a protein with an apparent molecular mass of 46 kDa. In CHO-K1 cells, the cDNA clone leads to the production of two major peptides with apparent molecular masses of 38 and 34 kDa, as determined by Western blotting with an antibody raised against a recombinant pssC protein. When CHO-K1 cells transfected with the cDNA clone are labelled with [35S]methionine for a short period, proteins immunoprecipitated with the antibody lack radioactive 38 and 34 kDa peptides, but contain two radioactive peptides with apparent molecular masses of 46 and 42 kDa instead. The pssC gene product predicted from the cDNA sequence has, near its C-terminus, a unique Leu-Gly-Ser-Thr sequence which is known as a processing site for Escherichia coli phosphatidylserine decarboxylase. A mutant pssC cDNA clone, in which Ser378 in the conserved sequence is replaced by Ala, leads to overproduction of 46, 42 and 38 kDa peptides, but not a 34 kDa peptide. This mutant clone is incapable of increasing phosphatidylserine decarboxylase activity, in contrast to the wild-type clone. These results indicate that the processing at the Leu-Gly-Ser-Thr sequence is essential for formation of the active enzyme. Thus, the pssC gene product is converted into mature phosphatidylserine decarboxylase through multiple steps of post-translational processing.
The gene G13 in the class III region of the human MHC encodes a potential DNA-binding protein
Authors: KHANNA Anu , CAMPBELL R. Duncan
Published: August 2015

G13 is a single-copy gene lying approx. 75 kb centromeric of the complement gene cluster in the class III region of the human MHC. The gene spans approx. 17 kb of DNA and has been shown to encode mRNA of approx. 2.7 kb that is present in cell lines representing lymphoid and non-lymphoid tissues, indicating that it is ubiquitously expressed. The complete nucleotide sequence of the 2.7 kb mRNA has been derived from cDNA and genomic clones. The longest open reading frame obtained for G13 codes for a 703 amino acid protein of approx. 77 kDa in molecular mass. Comparison of the putative G13 amino acid sequence with the protein databases revealed significant similarities with DNA-binding proteins of the leucine zipper class, including a human cAMP response element binding protein. G13 contains a bZIP motif, a region rich in basic amino acids adjacent to a coiled-coil leucine zipper domain, common to this class of proteins that is known to be involved in dimerization and DNA binding. Antibodies raised against a fragment encoding the C-terminal half of the putative G13 protein recognized a major polypeptide of approx. 86 kDa and a minor polypeptide of approx. 78 kDa on immunoblotting of U937 cell extracts; this has been confirmed by immunoprecipitation experiments. Even though it contained at least one potential bipartite nuclear localization signal, the G13 protein was present both in the cytoplasm and the nucleus of the fibroblast cells. Thus G13 might be a novel DNA-binding protein that is perhaps translocated to the nucleus in a regulated manner.
G-protein inhibition of phospholipase C-β1 in membranes: role of G-protein βγ subunits
Authors: LITOSCH Irene
Published: August 2015

Rat liver plasma membranes reconstituted with bovine brain phospholipase C β1 (PLC-β1) exhibit a dual regulation of PLC-β1 activity by G-proteins. Guanosine 5´-[γ-thio]triphosphate (GTP[S]; 0.1 nM) produced a 20-25% inhibition of PLC-β1 activity within 7 min of incubation. The addition of vasopressin resulted in near-basal levels of activity in the presence of 0.1 nM GTP[S]. Clonidine had little effect on the net inhibition due to GTP[S]. A similar antagonism between carbachol and GTP[S] occurred in cerebral cortical membranes containing endogenous PLC-β1 activity. αo/i-GDP (a mixture of GDP-liganded Goα and Giα) attenuated the GTP[S]-dependent inhibition of PLC-β1 whereas αo/i-GTP[S] had no effect, suggesting an involvement of G-protein βγ subunits in the inhibition of PLC-β1. Low concentrations of βγ subunits inhibited PLC-β1 activity. Inhibition was followed by reversal to basal activity and onset of stimulation as the βγ concentration was increased. Inhibition by βγ was dependent on the presence of membranes. These results indicate that G-protein βγ subunits constitute a mechanism by which G-proteins mediate a rapid and transient inhibition of PLC-β1.
High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization
Authors: ALI Stuart A. , JOAO Heidi C , CSONGA Robert , HAMMERSCHMID Franz , STEINKASSERER Alexander
Published: August 2015

Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% α-helix and 28% β-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.
The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes
Authors: GARRIGA Judit , MAYOL Xavier , GRAÑA Xavier
Published: August 2015

PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity. Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo.
Nitric oxide-induced apoptosis: p53-dependent and p53-independent signalling pathways
Authors: MESSMER Udo K , BRÜNE Bernhard
Published: August 2015

Nitric oxide (NO) generation initiates apoptotic cell death in different experimental systems. In RAW 264.7 macrophages the appearance of typical apoptotic markers is linked to inducible NO synthase induction. Mechanistically, accumulation of tumour suppressor p53 precedes apoptotic DNA fragmentation. With the use of S-nitroglutathione (GSNO) we correlated a dose-dependent p53 up-regulation to DNA fragmentation measured after 4 h and 8 h, respectively. Our studies revealed a linear correlation between the potency of five different NO donors with respect to apoptosis induction and p53 accumulation. Furthermore, we probed for NO-induced apoptosis after stable transfection of RAW 264.7 macrophages with plasmids encoding p53 antisense RNA. Clones with down-regulated p53 levels in response to GSNO exhibited a marked reduction in DNA fragmentation. Expression of the inducible NO synthase in response to lipopolysaccharide and interferon-γcaused apoptosis in RAW 264.7 macrophages and neomycin-vector controls within 24 h. In contrast, p53 antisense RNA-expressing clones appeared highly resistant towards endogenous NO, although inducible NO synthase induction with concomitant nitrite production remained unchanged. For RAW 264.7 macrophages our results established a functional role of the tumour suppressor p53 during NO-induced apoptotic cell death. However, p53 antisense experiments and the use of the p53-negative cell line U937 substantiated p53-independent signalling pathways operative during NO-mediated apoptosis.
Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart myoblastic cells
Authors: TRAN Khai , WONG Jason T , LEE Edmund , CHAN Alvin C. , CHOY Patrick C.
Published: August 2015

Cytosolic phospholipase A2 (cPLA2) selectively catalyses the release of arachidonic acid from the sn-2 position of glycerophospholipids to produce prostaglandins and leukotrienes. In this study, vitamin E enrichment of rat heart myoblastic H9c2 cells caused an increase in the release of arachidonate during ionophore (A23187) stimulation. PLA2 activity in the cytosolic fraction was also enhanced but enzyme activity in the particulate fraction was not affected by this treatment. Immunoblotting analysis with a polyclonal anti-cPLA2 antibody showed an increased level of the enzyme in vitamin E-treated cells. Direct incorporation of vitamin E into lipid vesicles in the assay mixture resulted in modulation of enzyme activity in a biphasic manner. Pretreatment of cells with phorbol 12-myristate 13-acetate, a known activator of protein kinase C, synergistically potentiated the ionophore-induced arachidonate release in both the control and vitamin E-treated cells. However, vitamin E treatment by itself did not affect the protein kinase C activity, indicating that the vitamin E-induced activation of cPLA2 was independent of the protein kinase C cascade. Collectively, these results suggest that vitamin E potentiates arachidonate release through the direct and/or indirect modulation of cPLA2 activity.
Isoforms of endoplasmic reticulum Ca2+-ATPase are differentially expressed in normal and diabetic islets of Langerhans
Authors: VÁRADI Anikó , MOLNÁR Elek , ÖSTENSON Claes-Göran , ASHCROFT Stephen J. H.
Published: August 2015

Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic β-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F β-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in β-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in the β-cell may contribute to the decreased insulin secretion seen in non-insulin dependent diabetes mellitus (NIDDM). We therefore investigated SERCA 2 and SERCA 3 mRNA expression by quantitative RT-PCR in islets prepared from Goto-Kakizaki (GK) rats, a non-obese spontaneous model of NIDDM. We found a significant reduction (about 68%) in SERCA 3 isoform expression. Since SERCA 2 expression was not significantly reduced, these genes are independently regulated and probably play distinct roles in islets of Langerhans. The marked decrease of SERCA 3 expression may constitute a defect in Ca2+ signalling in GK rat islets which could be a component of NIDDM.
Nucleosome core particles inhibit DNA triple helix formation
Authors: BROWN Philip M , FOX Keith R
Published: August 2015

We have used DNase I footprinting to examine the formation of DNA triple helices at target sites on DNA fragments that have been reconstituted with nucleosome core particles. We show that a 12 bp homopurine target site, located 45 bp from the end of the 160 bp tyrT(46A) fragment, cannot be targeted with either parallel (CT-containing) or antiparallel (GT-containing) triplex-forming oligonucleotides when reconstituted on to nucleosome core particles. Binding is not facilitated by the presence of a triplex-binding ligand. However, both parallel and antiparallel triplexes could be formed on a truncated DNA fragment in which the target site was located closer to the end of the DNA fragment. We suggest that intermolecular DNA triplexes can only be formed on those DNA regions that are less tightly associated with the protein core.
Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion
Authors: GREGORY Roland B , BARRITT Greg J
Published: August 2015

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro-Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+o-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trpl cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2–17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5´-[γ-thio]triphosphate (GTP[S]), guanosine 5´-[β,γ-imido]triphosphate (p[NH]ppG) and AlF4-, but not guanosine 5´-[β-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] or AlF4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.
Interleukin-13 is a potent activator of JAK3 and STAT6 in cells expressing interleukin-2 receptor-γ and interleukin-4 receptor-α
Authors: MALABARBA M. Grazia , RUI Hallgeir , DEUTSCH Harald H. J. , CHUNG Johanna , KALTHOFF Frank S. , FARRAR William L. , KIRKEN Robert A.
Published: August 2015

The lymphocyte growth factors interleukin-2 (IL2), IL4, IL7, IL9 and IL15 use the common IL2 receptor-γ (IL2Rγ) and activate the IL2Rγ-associated tyrosine kinase JAK3 (Janus kinase 3). IL13 is structurally related to IL4, competes with IL4 for binding to cell surface receptors and exhibits many similar biological effects. The molecular basis for this functional overlap between IL4 and IL13 has been attributed mainly to a shared use of the 140 kDa IL4Rα, since these cytokines appear to be uniquely different in that, according to several recent reports, IL13 does not recruit the IL2Rγ or JAK3. This notion has been supported by the identification of a novel 70 kDa IL13 receptor in certain IL13-responsive cell lines that lack IL2Rγ. The present study sheds new light on the issue of functional overlap between IL13 and IL4, by demonstrating for the first time that, in cells that express both IL2Rγ and IL4Rα, IL13 can mimic IL4-induced heterodimerization of IL2Rγ and IL4Rα, with consequent marked activation of JAK3 and the transcription factor STAT6 (IL4-STAT). Reconstitution experiments in BA/F3 cells showed that both cytokines require the simultaneous presence of IL4Rα and IL2Rγ to mediate JAK3 and proliferative responses, and analysis of 12 IL4Rα variants showed that IL4 and IL13 signals were equally affected by mutations of the cytoplasmic domain. We conclude that IL13 activates the IL2Rγ-associated JAK3 tyrosine kinase in appropriate cell types, and propose that IL13 is capable of interacting with multiple receptor subunits in a cell-dependent and combinatorial manner. Consequently, we predict that partial disruption of IL13 signal transduction also contributes to the severe combined immunodeficiency syndromes associated with inactivation of the IL2Rγ or JAK3 genes.
Identification of a novel opioid peptide (Tyr-Val-Pro-Phe-Pro) derived from human αS1 casein (αS1-casomorphin, and αS1-casomorphin amide)
Authors: KAMPA Marilenna , LOUKAS Spyros , HATZOGLOU Anastassia , MARTIN Patrice , MARTIN Pierre-Marie , CASTANAS Elias
Published: August 2015

A new casomorphin pentapeptide (αS1-casomorphin) has been isolated from the sequence of human αS1-casein [αS1-casein-(158–162)], with the sequence Tyr-Val-Pro-Phe-Pro. This peptide was found to bind with high affinity to all three subtypes of the κ-opioid receptor (κ1–κ3). When amidated at the C-terminus, αS1-casomorphin amide binds to the Δ- and κ3-opioid sites. Both αS1-casomorphin and its amide inhibit in a dose-dependent and reversible manner the proliferation of T47D human breast cancer cells. This anti-proliferative activity was greater for αS1-casomorphin, which was the most potent opioid in inhibiting T47D cell proliferation. In T47D breast cancer cells, other casomorphins have been found to bind to somatostatin receptors in addition to opioid sites. In contrast, αS1-casomorphin and its amide do not interact with somatostatin receptors in our system.
Ins(1,3,4,5)P4 is effective in mobilizing Ca2+ in mouse exocrine pancreatic acinar cells if phospholipase A2 is inhibited
Authors: ROWLES Sarah-Jayne , GALLACHER David V
Published: August 2015

In enzymically isolated mouse pancreatic acinar cells, under conditions of whole-cell patch-clamp current recording, the effect of phospholipase C-coupled agonists can be mimicked by internal perfusion of the intracellular second messenger Ins(1,4,5)P3 (10 µM) or its analogue Ins(2,4,5)P3 (10 µM). The inositol trisphosphates mimic receptor activation by releasing Ca2+ from intracellular stores and by promoting Ca2+ influx across the surface membrane. This Ca2+-mobilizing role of inositol polyphosphates seems to be confined to the inositol trisphosphates because internal perfusion of Ins(1,3,4,5)P4 (10 µM) is not associated with any Ca2+-dependent current activation. In this study we investigate the effects of 4-bromophenacyl bromide (4BPB), a putative inhibitor of phospholipase A2 and arachadonic acid production, on inositol polyphosphate-induced Ca2+ signalling. At 10 µM, 4BPB has no effect on unstimulated Ca2+-dependent membrane currents. However, if 4BPB is applied to cells internally perfused with 10 µM Ins(1,4,5)P3 or Ins(2,4,5)P3 then the current responses are rapidly potentiated. In cells internally perfused with 10 µM Ins(1,3,4,5)P4, which has itself no effect on membrane currents, application of 4BPB resulted in the activation of Ca2+-dependent currents, seen either as repetitive spikes of current or as sustained current activations. The application of arachidonic acid blocks the current responses evoked by the inositol trisphosphates and by Ins(1,3,4,5)P4/4BPB. These results suggest that in enzymically isolated pancreatic acinar cells phospholipase A2 activity is exerting an inhibitory effect on inositol polyphosphate-mediated Ca2+ mobilization. 4BPB removes this inhibition and potentiates the responses to internally perfused inositol trisphosphates and, importantly, makes 10 µM Ins(1,3,4,5)P4 as effective as 10 µM Ins(1,4,5)P3 in mobilizing intracellular Ca2+ and in promoting Ca2+ influx.
cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product
Authors: HALL Robert E. , AGARWAL Sujata , KESTLER Daniel P , COBB John A , GOLDSTEIN Keith M , CHANG Nan-Shan
Published: August 2015

P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line. It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-α and interleukin 6 in human monocytes and monocytic cell lines. The cDNA for P48 was cloned from Reh cellular RNA using 3´ reverse amplification of cDNA ends. Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells. Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated from Mycoplasma fermentans but not M. hominis, M. iowae, M. synoviae or M. lypophilum. Although initial studies using Mycoplasma culture and hybridization techniques had failed to reveal Mycoplasma infection in our Reh and Molt-4 cell lines, subsequent PCR studies using Mycoplasma genus-specific rRNA primers revealed Mycoplasma sequences in these cell lines. Using the P48 cDNA probe, we isolated a genomic clone from M. fermentans DNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium. The 5´ end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein. The P48 cDNA was expressed in pMAL in Escherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots. This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification. Consistent with this, Northern blot studies revealed a single 1 kb transcript. The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium. These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived from M. fermentans or a closely related species. P48 may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis.
Interleukin-2 induces γ-S-adenosyl-l-methionine synthetase gene expression during T-lymphocyte activation
Authors: TOBEÑA Rafael , HORIKAWA Saburo , CALVO Victor , ALEMANY Susana
Published: August 2015

The regulation of expression of the γ-S-adenosyl-L-methionine (AdoMet) synthetase gene was investigated in T-cells during G0/G1 transition, as well as throughout the G1 phase. Stimulation of G0 T-lymphocytes with concanavalin A induces AdoMet synthetase gene expression, starting 8 h after stimulation. Interleukin-2 (IL-2) stimulates the induction of this gene expression and AdoMet synthetase activity in G1 lymphoblasts, in part by an increase in the transcription rate of the gene. Phorbol esters, which also stimulate the proliferation of G1 lymphoblasts, show a similar kinetics of AdoMet synthetase mRNA induction. In contrast, the mRNA levels of the S-adenosyl-L-homocysteine hydrolase, another enzyme of the methionine cycle, remain unchanged upon IL-2 or phorbol 12,13-dibutyrate treatment. Dexamethasone and 8Br-cAMP, both inhibitors of lymphocyte proliferation, are able to block the expression of the AdoMet synthetase gene and, consequently, AdoMet synthetase activity. Together these findings indicate that the AdoMet synthetase gene is subject to cell-cycle regulation in T-lymphocytes
Characterization of two forms of protein kinase C α, with different substrate specificities, from skeletal muscle
Authors: SCHMITZ-PEIFFER Carsten , BROWNE Carol L , BIDEN Trevor J
Published: August 2015

We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the presence of two major activities. These did not correspond to different PKC isoenzymes but seemed to represent two species of PKC α as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC α; detection of the two species by three specific but unrelated anti-(PKC α) antibodies; immunodepletion of both activities with anti-(PKC α) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid for activation. These species, termed PKC α1 and PKC α2, phosphorylated the modified conventional PKC pseudosubstrate peptide (19–31, Ser-25) equally well. Importantly, however, the activities differed in that PKC α1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC α2. Similarly, incubation of crude muscle extracts with either PKC α1 or α2 gave rise to different protein phosphorylation patterns. The involvement of proteolysis, dephosphorylation or oxidative modification in the interconversion of PKC α1 and PKC α2 during preparation was ruled out. Although some PKC-binding proteins were detected in overlay assays, their presence did not explain the anomalous PKC α2 activity. The results suggest that a modification of PKC α in situ limits its substrate specificity, and indicate an additional level of control of the kinase that may be a site for modulation of PKC-mediated signal transduction.
Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse δ opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation
Authors: BURT Andrew R , CARR I. Craig , MULLANEY Ian , ANDERSON Neil G. , MILLIGAN Graeme
Published: August 2015

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse Δ opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5´-[γ-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8–1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxin-sensitive manner, but both the kinetics and the concentration–response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a Gi-dependent manner, and show that only a very small proportion of the cellular Gi population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.
Role for L-selectin in lipopolysaccharide-induced activation of neutrophils
Authors: MALHOTRA Rajneesh , PRIEST Richard , BIRD Michael I.
Published: August 2015

The activation of leucocytes by bacterial cell wall lipopolysaccharide (LPS) contributes to the pathogenesis of septic shock. LPS is known to interact with several cell-surface proteins, including CD14, when presented as a complex with serum LPS-binding protein. However, the identity of the receptor responsible for LPS signalling and leucocyte activation is unknown. Interestingly, mice deficient in cell-surface L-selectin were dramatically resistant to the lethal effects of high doses of LPS in a model of septic shock. Recently we reported that L-selectin binds to cardiolipin and other charged phospholipids at a site distinct from the carbohydrate-binding site. Structural similarities between charged phospholipids and the lipid A moiety of LPS prompted us to investigate interactions between L-selectin and LPS. Herein we show that L-selectin is a neutrophil surface receptor for LPS and lipotechoic acid. The binding of LPS to L-selectin is independent of serum and Ca2+, and is blocked by antibodies to L-selectin and fucoidan. Furthermore, the interaction of LPS with cell-surface L-selectin results in superoxide production, indicating that L-selectin can mediate both binding and activation of human neutrophils. These findings suggest novel therapeutic approaches for the treatment of septic shock.
Surface properties, morphology and protein composition of pulmonary surfactant subtypes
Authors: PUTMAN Esther , CREUWELS Lambert A. J. M. , van GOLDE Lambert M. G. , HAAGSMAN Henk P.
Published: August 2015

Separation of surfactant subtypes is now commonly used as a parameter in assessing the amount of active compared with inactive material in various models of lung injury. The protein content, morphology and surface activity were determined of the heavy and light subtype isolated by differential centrifugation. Here we report the presence of surfactant proteins B and C in the heavy subtype but not in the light subtype. Adsorption studies revealed that separation of fast adsorbing bronchoalveolar lavage resulted in slowly adsorbing heavy and light subtypes. Surfactant, reconstituted from heavy and light fractions, did not show a high adsorption rate. It is concluded that the isolation procedures might result in a loss of fast adsorbing surfactant structures. Surface area cycling was used as a model in vitro for the extracellular surfactant metabolism. The heavy subtype is converted into the light subtype during conversion. Conversion performed with resuspended heavy subtype revealed the generation of a disparate subtype. Furthermore it was found that the conversion was dependent on preparation and handling of the samples before cycling. Finally, adsorption studies at low surfactant concentrations revealed a delayed adsorption of lipid-extracted surfactants compared with natural surfactants. These observations emphasize the importance of the (surfactant-associated protein A-dependent) structural organization of surfactant lipids in the adsorption process.
Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2Cα
Authors: MARLEY Anna E. , SULLIVAN Jane E. , CARLING David , ABBOTT W. Mark , SMITH Graeme J. , TAYLOR Ian W. F. , CAREY Frank , BERI Raj K.
Published: August 2015

The use of protein phosphatase inhibitors has been instrumental in defining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B. Identification of the role of PP2C in vivo has been hampered, in part, by the unavailability of specific inhibitors. In order to facilitate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further characterize this enzyme at the molecular level by site-directed mutagenesis and X-ray crystallography, we have expressed active recombinant human PP2Cα (rPP2Cα) in Escherichia coli. Biochemical characterization of rPP2Cα showed that it could hydrolyse p-nitrophenyl phosphate (pNPP) although, in contrast with native PP2C, this was not stimulated by Mg2+. As with native PP2C, okadaic acid failed to inhibit rPP2Cα, whereas 50 mM NaF dramatically inhibited its activity. An alignment of the amino acid sequence of AMP-activated protein kinase (AMPK) with those of other serine/threonine protein kinases around the regulatory phosphorylation site (subdomains VII–VIII) revealed a high degree of conservation. Phosphopeptides derived from this region of AMPK and containing the almost invariant threonine (Thr172 in AMPK) were found to be good substrates for rPP2Cα. We also showed that rPP2Cα can inactivate AMPK, but only in the presence of Mg2+. To define the regions of PP2Cα important for catalytic activity, we expressed a number of truncated proteins based on the sequence and proposed domain structure of the PP2Cα homologue from Paramecium tetraurelia. Deletion of 75 residues (9 kDa) from the C-terminus appeared to have little effect on the catalytic activity using pNPP, phosphopeptides or AMPK as substrates. This suggests that the residues important in catalysis lie elsewhere in the protein. A further deletion of the C-terminus led to a completely inactive and very poorly soluble protein.
Lactoferrin regulates the activity of heparin proteoglycan-bound mast cell chymase: characterization of the binding of heparin to lactoferrin
Authors: PEJLER Gunnar
Published: August 2015

Rat mast cell protease 1 (RMCP-1) is a secretory granule serine protease (chymase) that is recovered in vivo in a macromolecular complex with heparin proteoglycan (PG). We have previously shown that heparin activates RMCP-1 and that RMCP-1, when bound to heparin PG, is largely resistant to inhibition by a variety of macromolecular protease inhibitors. In the search for alternative mechanisms in the regulation of RMCP-1 activity, we hypothesized that heparin antagonists, by interfering with the RMCP-1/heparin PG interaction, might influence the activity of heparin-bound mast cell chymase. In the present study, lactoferrin (LF), a heparin-binding protein, was assessed for RMCP-1 inhibiting activity. LF proved to decrease the activity of heparin PG-associated RMCP-1, although a portion of the enzyme activity was resistant to regulation. The mechanism of regulation was shown to involve the displacement of RMCP-1 from heparin PG, and LF caused an approx. 6-fold increase in the apparent Km of the RMCP-1–heparin PG complex for the chromogenic substrate S-2586. The interaction of LF with heparin was characterized. Pig mucosal heparin and endogenous heparin PG were equally effective in binding LF, whereas heparan sulphate bound with lower affinity. None of dermatan sulphate, chondroitin sulphate or hyaluronan were effective in binding LF. Further, the 6-O-, 2-O- and N-sulphate groups in heparin were of approximately equal importance for binding. Octasaccharides were the smallest heparin oligosaccharides showing significant binding to LF.
Influence of metal ions on substrate binding and catalytic activity of mammalian protein geranylgeranyltransferase type-I
Authors: ZHANG Fang L. , CASEY Patrick J.
Published: August 2015

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group from the prenyl donor geranylgeranyl diphosphate (GGPP) to the cysteine residue of substrate proteins containing a C-terminal CaaX-motif (a sequence motif of proteins consisting of an invariant Cys residue fourth from the C-terminus). The GGTase-I heterodimer contains one atom of zinc, and this metal is required for enzyme activity. In this regard, GGTase-I is similar to the related enzyme protein farnesyltransferase (FTase); the latter enzyme also requires Mg2+ for activity. The current studies were undertaken in an attempt to explore further the role of bivalent metal ions in the activity of GGTase-I. Surprisingly, we found that GGTase-I and FTase have different metal requirements. Specifically, in marked contrast to FTase, GGTase-I does not require Mg2+ for activity. Direct binding assays, including a novel fluorescence-based technique, were employed to obtain quantitative information on the interaction of substrates with GGTase-I. Using these assays, we demonstrate that the Zn2+ in GGTase-I is required for peptide, but not for isoprenoid, substrate binding. Moreover, binding of GGPP protects GGTase-I from inactivation by zinc-chelating reagents; this protective effect is not seen with binding of peptide substrates. Metal substitution studies show that the Zn2+ in GGTase-I can be replaced by Cd2+, and that the Cd form of GGTase-I has altered specificity with regard to utilization of both peptide and isoprenoid substrates. The significance of these findings in relation to proposed mechanisms for the GGTase-I reaction is discussed.
Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines
Authors: POOLEY Linda , SHAKUR Yasmin , RENA Graham , HOUSLAY Miles D.
Published: August 2015

Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3–4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.
Human tissue non-specific alkaline phosphatases: sugar-moiety-induced enzymic and antigenic modulations and genetic aspects
Authors: NOSJEAN Olivier , KOYAMA Iwao , GOSEKI Masae , ROUX Bernard , KOMODA Tsugikazu
Published: August 2015

To investigate the possible role(s) of glycans in human tissue non-specific alkaline phosphatase (TNAP) activity, the isoenzymes were purified and treated with various exo- and endo-glycosidases. Catalytic activity, oligomerization, conformation and immunoreactivity of the modified TNAPs were evaluated. All TNAPs proved to be N-glycosylated, and only the liver isoform (LAP) is not O-glycosylated. Usually, the kidney (KAP) and bone (BAP) isoenzymes are similar and cannot be clearly discriminated. Differences between the immunoreactivity of KAP/BAP and LAP with a BAP antibody were mainly attributed to the N-glycosylated moieties of the TNAPs. In addition, elimination of O-glycosylations moderately affects the TNAP reactivity. Interestingly, N-glycosylation is absolutely essential for TNAP activity, but not for that of the placental or intestinal enzymes. According to the deduced amino acid sequence of TNAP cDNA, Asn-213 is a possible N-glycosylation site, and our present findings suggest that this sugar chain plays a key role in enzyme regulation. With regard to the oligomeric state of alkaline phosphatase (AP) isoforms, the dimer/tetramer equilibrium is dependent on the deglycosylation of glycosyl-phosphatidylinositol(GPI)-free APs, but not GPI-linked APs. This equilibrium does not affect the AP conformation as observed with CD. With regard to TNAPs, no data were available on the gene expression or nature of the 5ƀ-non-translated leader exon of human KAP, as opposed to BAP and LAP genes. cDNA sequencing revealed that cortex/medulla KAP is genetically related to BAP, and medulla KAP to LAP.
Ras activation in platelets after stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C
Authors: SHOCK David D. , HE Kai , WENCEL-DRAKE June D. , PARISE Leslie V.
Published: August 2015

Several reports have indicated that the small G-protein Ras is not present immunologically in platelets. However, here we report the identification of Ras in platelets by immunoprecipitation with the Ras-specific monoclonal antibodies Y13-259 or Y13-238, followed by Western blotting. The presence of Ras was not due to contamination of samples with erythrocytes or leucocytes. Immunofluorescence studies indicated that Ras was present in a peripheral rim pattern in fixed, permeabilized platelets, suggesting an intracellular, plasma membrane location. Activation of platelets with the thrombin receptor peptide42Ő50, the prostaglandin H2/thromboxane A2 mimetic U46619 or phorbol 12-myristate 13-acetate induced a rapid increase in GTP-bound, activated Ras. In each case, this increase was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide GF 109203X, suggesting that Ras is activated downstream of PKC in platelets. Thus the activation of Ras in platelets by agonists will now allow consideration of multiple potential Ras-dependent signal transduction pathways in platelet activation processes.