bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulated and unregulated pathways for MUC2 mucin secretion in human colonic LS180 adenocarcinoma cells are distinct
Authors: McCool D J , Forstner J F , Forstner G G
Published: August 2015

We have shown previously [McCool, Forstner and Forstner (1994) Biochem. J. 302, 111-118] using pulse-chase labelling of mucin with [3H]threonine that LS180 colonic tumour cells synthesize and secrete MUC2 without the addition of secretagogues. Treatment of the LS180 cells with monensin to disrupt Golgi function was also found to inhibit baseline secretion almost completely. In this paper we show that addition of nocodazole to inhibit microtubule assembly reduced baseline secretion by 53% over a 6 h chase period. In contrast, cytochalasin D did not affect the rate of unstimulated mucin synthesis or secretion, suggesting that baseline secretion is not influenced by disruption of actin microfilaments. In addition, regulated mucin secretion by LS180 cells was studied in response to carbachol, phorbol 12-myristate 13-acetate and A23187. Mucin released in response to secretagogues behaved identically on SDS/PAGE to that secreted under baseline conditions. T84 cells and the B6 subclone of the HT29 cell line responded in a similar manner to LS180 cells and secreted high-molecular-mass mucin which included MUC2 and behaved like LS180 mucin on SDS/PAGE. Neither monensin nor nocodazole significantly affected secretagogue-stimulated mucin secretion. Since these compounds inhibited secretion of labelled mucin under baseline conditions, mucin released by secretagogues must have come from a separate, unlabelled mucin pool in stored granules. Cytochalasin D, on the other hand, caused the release of small amounts of stored mucin, suggesting that actin microfilaments participate in regulated exocytosis. Thus two kinds of mucin secretion occur in LS180 cells. Unregulated secretion depends upon continuous transport of mucin granules from Golgi vesicles to the cell surface and does not utilize stored mucin, whereas regulated secretion involves the release of mucin from storage granules and is not affected by microtubule or Golgi disruption.
Design of a new protease inhibitor by the manipulation of the bait region of α2-macroglobulin: inhibition of the tobacco etch virus protease by mutant α2-macroglobulin
Authors: Van Rompaey L , Proost P , Van den Berghe H , Marynen P
Published: August 2015

Human alpha 2-macroglobulin (alpha 2M) inhibits a broad spectrum of proteases by changing its conformation and physically confining the enzyme. The inhibitory spectrum of alpha 2M is defined by a stretch of 39 amino acids, the bait region, located near the middle of the alpha 2M monomers. To investigate whether a new inhibitory specificity can be introduced by the manipulation of the bait region, recombinant alpha 2M (r alpha 2M) was produced in which the primary cleavage site was replaced by a heptapeptide containing the cleavage specificity of tobacco etch virus (TEV) protease. This protease is not inhibited by wild-type alpha 2M. The r alpha 2M, produced in an episomal expression system, was fully functional and able to inhibit the tobacco etch virus protease according to its normal ‘trap’ mechanism. The manipulation of the bait region of alpha 2M thus allows the design of new, specific protease inhibitors.
Stimulation of phosphatidylcholine biosynthesis in mouse MLE-12 type-II cells by conditioned medium from cortisol-treated rat fetal lung fibroblasts
Authors: MacDonald J I S , Possmayer F
Published: August 2015

Treatment of murine adult MLE-12 type-II and fetal-rat type-II cells with fetal-rat-fibroblast-conditioned medium (FFCM) resulted in a 2-fold stimulation of [14C]choline incorporation into phosphatidylcholine. Soluble CTP:phosphocholine cytidylyltransferase (CT) activity was increased approx. 3-fold in FFCM-treated fetal-rat type-II cells but was not changed in MLE-12 cells. Neither choline kinase nor cholinephosphotransferase activities were affected by treatment of MLE-12 cells with FFCM. Long-term labelling of MLE-12 cells with [14C]choline, followed by a 14-18 h chase with FFCM, resulted in a 2.5-fold decrease in [14C]phosphocholine levels relative to controls, suggesting that CT was being activated. In contrast, oleate treatment increased CT activity in the particulate fraction in both cells. Western blots indicate that soluble CT undergoes dephosphorylation in response to FFCM, but no translocation to the particulate fraction was noted. Treatment with oleate stimulated a marked translocation. Tryptic phosphopeptide maps from FFCM-treated cells revealed only minor alterations in the phosphorylation pattern. It is concluded that FFCM and oleate activate CT through different mechanisms. The results are consistent with FFCM activating CT in MLE-12 as well as fetal type-II cells. However, the reason why this activation cannot be detected in vitro is not known.
Altering kinetic mechanism and enzyme stability by mutagenesis of the dimer interface of glutathione reductase
Authors: Bashir A , Perham R N , Scrutton N S , Berry A
Published: August 2015

In wild-type glutathione reductase from Escherichia coli residues Val421 and Ala422 are located in an alpha-helix in a densely packed and hydrophobic region of the dimer interface, with their side chains packed against those of residues Ala422′ and Val421′ in the second subunit. A series of mutant glutathione reductases was constructed in which the identities of the residues at positions 421 and 422 were changed. Mutations were designed so as to present like charges (mutants Val421-->Glu:Ala422-->Glu and Val421-->Lys:Ala422-->Lys) or opposite charges (mutant Val421-->Lys:Ala422-->Glu) across the dimer interface to assess the role of electrostatic interactions in dimer stability. A fourth mutant (Val421-->His:Ala422-->His) was also constructed to investigate the effects of introducing a potentially protonatable bulky side chain into a crowded region of the dimer interface. In all cases, an active dimeric enzyme was found to be assembled but each mutant protein was thermally destabilized. A detailed steady-state kinetic analysis indicated that each mutant enzyme no longer displayed the Ping Pong kinetic behaviour associated with the wild-type enzyme but exhibited what was best described as a random bireactant ternary complex mechanism. This leads, depending on the chosen substrate concentration, to apparent sigmoidal, hyperbolic or complex kinetic behaviour. These experiments, together with others reported previously, indicate that simple mutagenic changes in regions distant from the active site can lead to dramatic switches in steady-state kinetic mechanism.
Differences in the binding of transforming growth factor β1 to the acute-phase reactant and constitutively synthesized α-macroglobulins of rat
Authors: Webb D J , Crookston K P , Figler N L , Lamarre J , Gonias S L
Published: August 2015

Human alpha 2-macroglobulin (alpha 2M) is a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor beta 1 (TGF-beta 1). The constitutively synthesized homologue of human alpha 2M in the adult rat is alpha 1M. Rat alpha 2M is an acute-phase reactant, expressed at high levels in experimental trauma, pregnancy and in certain pathological conditions. The physiological role of rat alpha 2M is not known. In this investigation, we demonstrated that rat alpha 1M and rat alpha 2M bind TGF-beta 1. The equilibrium dissociation constants (KD) for the binding of TGF-beta 1 to the native forms of alpha 1M and alpha 2M were 257 and 109 nM respectively. alpha 1M underwent conformational change when it reacted with methylamine. The resulting product bound TGF-beta 1 with higher affinity (32 nM). Methylamine-treated rat alpha 2M did not undergo conformational change and did not bind TGF-beta 1 with increased affinity. Previous studies suggest that the native conformation may be the principal form responsible for the cytokine-carrier activity of alpha 2M in plasma and serum-supplemented cell culture medium. To confirm that native rat alpha 2M is a more efficient TGF-beta 1 carrier than native alpha 1M, fetal bovine heart endothelial cell (FBHE) proliferation assays were performed. TGF-beta 1 (5 pM) inhibited FBHE proliferation, and native alpha 2M (0.3 microM) counteracted this activity whereas alpha 1M (0.3 microM) had almost no effect. Rat alpha 2M underwent conformational change when it reacted with plasmin incorporating 1.1 mol of plasmin/mol. alpha 2M-plasmin bound TGF-beta 1; the KD (61 nM) was lower (P < 0.01) than that determined for the native alpha 2M-TGF-beta 1 interaction. These studies demonstrate that both rat alpha-macroglobulins are carriers of TGF-beta 1. The native form of rat alpha 2M probably has a predominant role, compared with native alpha 1M, as a TGF-beta 1 carrier in the plasma during the acute-phase response.
Ethanol has different effects on Ca2+-transport ATPases of muscle, brain and blood platelets
Authors: Mitidieri F , de Meis L
Published: August 2015

The effects of ethanol on different sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCAs) were studied. In sarcoplasmic reticulum vesicles, ethanol concentrations varying from 5 to 20% promoted a progressive inhibition of Ca2+ uptake, enhancement of Ca2+ efflux, activation of the ATPase activity, increase of the enzyme phosphorylation by ATP and inhibition of enzyme phosphorylation by P1. The effects of ethanol on Ca2+ uptake and Ca2+ efflux were antagonized by Mg2+, P(i) and spermine. The increased efflux promoted by ethanol was antagonized by Ca2+ and thapsigargin. In brain and platelet vesicles a biphasic effect of ethanol was observed, so that activation occurred at low concentrations (5-10%) and inhibition at higher concentrations. The activation was not observed with the use of n-propanol and n-butanol. Different from the situation in sarcoplasmic reticulum, the decrease of the Ca2+ uptake in brain and platelet vesicles was associated with an inhibition of the ATPase activity. Mg2+ and P(i) antagonized the enhancement of Ca2+ efflux and the inhibition of Ca2+ uptake promoted by ethanol. However, thapsigargin and Ca2+ did not arrest the Ca2+ efflux promoted by ethanol in brain and platelet preparations. These results suggest that, in sarcoplasmic reticulum vesicles, ethanol uncouples the pump, promoting its activity as a Ca2+ channel. The SERCA isoform found in skeletal muscle has different properties from the isoforms found in brain and blood platelets.
Evidence for receptor and G-protein regulation of a phosphatidylethanolamine-hydrolysing phospholipase A1 in guinea-pig heart microsomes: stimulation of phospholipase A1 activity by DL-isoprenaline and guanine nucleotides
Authors: Badiani K , Arthur G
Published: August 2015

While evidence has been presented for the receptor-mediated activation of phospholipases A2, C and D, the activation of phospholipase A1 subsequent to receptor activation has not been established. Phospholipase A1-catalysed hydrolysis of 1-palmitoyl-2-linoleoyl-glycerophosphoethanolamine (GPE) by guinea-pig heart microsomes was stimulated 40-60% by isoprenaline. This isoprenaline-mediated increase in activity was blocked by propranolol and butoxamine, a specific beta 2-adrenergic antagonist, but not by atenolol, a specific beta 1-adrenergic antagonist. Neither clonidine nor phenylephrine, alpha 1- and alpha 2-adrenergic agonists respectively, had a stimulatory effect on the hydrolysis of the PE substrate. Guanosine 5′(-)[gamma-thio]triphosphate (GTP[S]) and guanosine 5′(-)[beta, gamma-imido]triphosphate, but not guanosine 5′(-)[beta-thio]diphosphate (GDP[S]) or adenosine 5′(-)[gamma-thio]triphosphate, stimulated the hydrolysis of 1-palmitoyl-2-linoleoyl-GPE by phospholipase A1. GDP[S] inhibited the isoprenaline-mediated stimulation of phospholipase A1 activity. Phospholipase A1 hydrolysis of 1-palmitoyl-2-linoleoyl-GPE was not dependent on cations; however, the stimulatory effects of isoprenaline and GTP[S] on the hydrolytic activity were abolished by cation chelators. The above data suggest that phospholipase A1 activity in guinea-pig heart microsomes is activated by the binding of isoprenaline to beta 2-adrenergic receptors. Furthermore the stimulation of phospholipase A1 activity by the agonist may be mediated via activation of G-proteins.
Biosynthesis of recombinant human pro-α1(III) chains in a baculovirus expression system: production of disulphide-bonded and non-disulphide-bonded species containing full-length triple helices
Authors: Tomita M , Ohkura N , Ito M , Kato T , Royce P M , Kitajima T
Published: August 2015

We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the pro-alpha1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displayed a melting temperature (Tm) of approx. 38 degrees C as assessed by its resistance to digestion by a mixture of trypsin and chymotrypsin, slightly lower than that of 39.5 degrees C for procollagen III synthesized by cultured human dermal fibroblasts, and reflected a slight degree of under-hydroxylation of prolyl residues. This is possibly a consequence of the lower incubation temperature of insect cells, or of an insufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of similar thermal stability containing an apparently full-length triple-helical region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the system promises to be useful in the study of procollagen chain association and subsequent folding.
The 4F2hc surface antigen is necessary for expression of system L-like neutral amino acid-transport activity in C6-BU-1 rat glioma cells: evidence from expression studies in Xenopus laevis oocytes
Authors: Bröer S , Bröer A , Hamprecht B
Published: August 2015

Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems in all non-epithelial cells. Its molecular structure is not known. To clone the neutral amino acid-transporter system L, we followed an expression cloning strategy using Xenopus laevis oocytes. A cDNA library derived from C6-BU-1 rat glioma cells was used as a source, because high expression of system L activity could be demonstrated with polyadenylated RNA isolated from these cells, when injected into Xenopus laevis oocytes [Bröer, Bröer and Hamprecht (1994) Biochim. Biophys. Acta 1192, 95-100]. A single clone (ILAT) was identified, the sense cRNA of which, on injection into Xenopus laevis oocytes, stimulated sodium-independent isoleucine transport by about 100-fold. Further characterization revealed that transport of cationic amino acids was also stimulated. Sequencing of the cDNA showed that the identified clone is the heavy chain of the rat 4F2 surface antigen, a marker of tumour cells and activated lymphocytes. Uptake of neutral and cationic amino acids was not stimulated by the presence of Na+ ions. Antisense cRNA transcribed from this clone or antisense oligonucleotides, when co-injected with polyadenylated RNA from C6-BU-1 rat glioma cells, completely suppressed system L-like isoleucine-transport activity. We conclude that ILAT is necessary for expression of system L-like amino acid-transport activity by polyadenylated RNA from C6-BU-1 rat glioma cells.
Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs
Authors: Perez-Terzic C M , Chini E N , Shen S S , Dousa T P , Clapham D E
Published: August 2015

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.
Expression of human dopamine β-hydroxylase in Drosophila Schneider 2 cells
Authors: LI Bin , TSING Stan , KOSAKA Alan H. , NGUYEN Binh , OSEN Eric G. , BACH Chinh , CHAN Hardy , BARNETT Jim
Published: August 2015

Human dopamine β-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The Km of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.
A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent β cells
Authors: KOWLURU Anjaneyulu , SEAVEY Scott E. , RHODES Christopher J. , METZ Stewart A.
Published: August 2015

Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the β subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the β cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [γ-32P]ATP or [γ-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an antiserum directed against the common β subunit of trimeric G-proteins. Disruption of the αβγ trimer (by pretreatment with either fluoroaluminate or guanosine 5ʹ-[γ-thio]triphosphate) prevented β subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure β subunit alone or in combination with the exogenous purified α subunit of transducin did not result in the phosphorylation of the β subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for β subunit phosphorylation. Incubation of phosphorylated β subunit with Gα·GDP accelerated the dephosphorylation of the β subunit, accompanied by the formation of Gα·GTP. Immunoblotting detected multiple α subunits (of Gi, Go and Gq) and at least one β subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in β cells which contrasts with the classical receptor-agonist mechanism: Gβ undergoes transient phosphorylation at a histidine residue by a GTP-specific protein kinase; this phosphate, in turn, may be transferred via a classical Ping-Pong mechanism to Gα·GDP (inactive), yielding the active configuration Gα·GTP in secretory granules (a strategic location to modulate exocytosis).
Sphingomyelinase stimulates 2-deoxyglucose uptake by skeletal muscle
Authors: TURINSKY Jiri , NAGEL G. William , ELMENDORF Jeffrey S. , DAMRAU-ABNEY Alice , SMITH Terry R.
Published: August 2015

The effects of sphingomyelinase, phosphorylcholine, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide) and sphingosine on basal and insulin-stimulated cellular accumulation of 2-deoxy-D-glucose in rat soleus muscles were investigated. Preincubation of muscles with sphingomyelinase (100 or 200 m-units/ml) for 1 or 2 h augmented basal 2-deoxyglucose uptake by 29-91%, and that at 0.1 and 1.0 m-unit of insulin/ml by 32-82% and 19-25% respectively compared with control muscles studied at the same insulin concentrations. The sphingomyelinase-induced increase in basal and insulin-stimulated 2-deoxyglucose uptake was inhibited by 91% by 70 μM cytochalasin B, suggesting that it involves glucose transporters. Sphingomyelinase had no effect on the cellular accumulation of L-glucose, which is not transported by glucose transporters. The sphingomyelinase-induced increase in 2-deoxyglucose uptake could not be reproduced by preincubating the muscles with 50 μM phosphorylcholine, 50 μM C2-ceramide or 50 μM C6-ceramide. Preincubation of muscles with 50 μM sphingosine augmented basal 2-deoxyglucose transport by 32%, but reduced the response to 0.1 and 1.0 m-unit of insulin/ml by 17 and 27% respectively. The stimulatory effect of sphingomyelinase on basal and insulin-induced 2-deoxyglucose uptake was not influenced by either removal of Ca2+ from the incubation medium or dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum. This demonstrates that Ca2+ does not mediate the action of sphingomyelinase on 2-deoxyglucose uptake. Sphingomyelinase also had no effect on basal and insulin-stimulated activities of insulin receptor tyrosine kinase and phosphatidylinositol 3-kinase. In addition, 1 and 5 μM wortmannin, an inhibitor of phosphatidylinositol 3-kinase, failed to inhibit the sphingomyelinase-induced increase in 2-deoxyglucose uptake. These results suggest that sphingomyelinase does not increase 2-deoxyglucose uptake by stimulating the insulin receptor or the initial steps of the insulin-transduction pathway. The data suggest the possibility that sphingomyelinase increases basal and insulin-stimulated 2-deoxyglucose uptake in skeletal muscle as the result of an unknown post-receptor effect.
Comparison of lipid aggregation and self-aggregation activities of pulmonary surfactant-associated protein A
Authors: RUANO Miguel L. F. , MIGUEL Eugenio , PEREZ-GIL Jesus , CASALS Cristina
Published: August 2015

1. We compared the Ca2+ dependence of the self-aggregation of surfactant protein A (SP-A) with that of vesicle aggregation induced by SP-A. The Ca2+ concentration required for half-maximal activity of lipid aggregation was 0.74±0.29 μM (n = 4) for pig SP-A and 98±5 μM (n = 2) for dog SP-A. In contrast, the threshold concentration of Ca2+ required to induce self-association of both pig and dog SP-A was 0.5 mM. The Ca2+ concentration needed for half-maximal self-association was 2.36±0.15 mM (n = 4) and 0.70±0.06 mM (n = 2) for pig and dog SP-A respectively. 2. We also compared the effect of Ca2+ on the trypsin sensitivity of lipid-free and membrane-bound SP-A. At 1 μM Ca2+, the tryptic digestion patterns of dog and pig lipid-free SP-A were quite different. Dog SP-A was very sensitive to proteolysis, being almost completely digested by 30 min, while pig SP-A was very resistant, even after 12 h. After protein aggregation of lipid-free SP-A (at 5 mM Ca2+), the accessibility of the trypsin cleavage targets of the protein depended on the SP-A species (self-aggregated pig SP-A became more sensitive to degradation than its non-aggregated form, whereas self-aggregated dog SP-A was less susceptible). In contrast, membrane-bound SP-A, from either pig or dog, was clearly protected from trypsin degradation at both low (1 μM) or high (1 mM) Ca2+ concentrations. The protection was slightly higher at 1 mM Ca2+ when the extent of lipid/SP-A aggregates was maximal. 3. On the other hand, vesicle aggregation activity of SP-A was decreased by 30-40% by removing the oligosaccharide moiety of the protein, whereas self-aggregation was not influenced by deglycosylation. The presence of mannan (at concentrations not lower than 10 μg/μl) decreased vesicle aggregation induced by dog and pig SP-A by a mechanism that is independent of the binding of mannan to the carbohydrate-binding domain of SP-A. Self-aggregation of SP-A was not affected by the presence of sugars. 4. From these results, we conclude that: (1) the process of lipid aggregation induced by SP-A cannot be correlated with that of self-association of the protein occurring at supramillimolar concentrations of Ca2+; and (2) the N-linked carbohydrate moiety of SP-A and the ability of SP-A to bind carbohydrates are not involved in lipid aggregation.
Identification of a transcription factor that binds to the S box of the I-A β gene of the major histocompatibility complex
Authors: CELADA Antonio , GIL Pilar , McKERCHER Scott R. , MAKI Richard A.
Published: August 2015

Class II genes of the MHC show a striking homology upstream of the transcription start site that is composed of three conserved sequences (S, X and Y boxes, each separated by 15–20 bp). The presence of the S-box sequence in the mouse MHC class II gene I-Aβ was examined for its influence on the expression of this gene. Deletion or mutation of the S box decreased the induction of chloramphenicol acetyltransferase (CAT) activity in B lymphocytes by 32%. In macrophages, deletion or mutation of the S box abolished interferon-γ (IFN-γ) inducibility of CAT activity. Using a gel-retardation assay, we have identified a nuclear factor whose binding site overlaps the 7-mer conserved sequence of the S box. This factor is present in lymphocytes, macrophages, mastocytes and fibroblasts. Surprisingly, binding of this nuclear factor to DNA was induced by IFN-γ in bone-marrow-derived macrophages, but not in macrophage-like cell lines. The binding site for this factor was defined by DNase I footprinting and partially purified by using an affinity column containing double-stranded oligonucleotides containing a sequence of the S box. A prominent protein of 43 kDa was found that bound specifically to the S-box sequence.
Kinetics and thermodynamics of the binding of riboflavin, riboflavin 5′-phosphate and riboflavin 3′,5′-bisphosphate by apoflavodoxins
Authors: PUEYO José J. , CURLEY Paul G. , MAYHEW Stephen G.
Published: August 2015

The reactions of excess apoflavodoxin from Desulfovibrio vulgaris, Anabaena variabilis and Azotobacter vinelandii with riboflavin 5´-phosphate (FMN), riboflavin 3´,5´-bisphosphate and riboflavin are pseudo-first-order. The rates increase with decreasing pH in the range pH 5–8, and, in general, they increase with increasing ionic strength to approach a maximum at an ionic strength greater than 0.4 M. The rate of FMN binding in phosphate at high pH increases to a maximum at an ionic strength of about 0.1 M, and then decreases as the phosphate concentration is increased further. The dissociation constants for the complexes with FMN and riboflavin decrease with an increase of ionic strength. Inorganic phosphate stabilizes the complex with riboflavin. The effects of phosphate on riboflavin binding suggest that phosphate interacts with the apoprotein at the site normally occupied by the phosphate of FMN. Redox potentials determined for the oxidized/semiquinone and semiquinone/ hydroquinone couples of the riboflavin and FMN complexes were used with Kd values for the complexes with the oxidized flavins to calculate values for Kd for the semiquinone and hydroquinone complexes. The hydroquinone complexes are all less stable than the complexes with the two other redox forms of the flavin. Destabilization of the hydroquinone is less marked in the complexes with riboflavin, supporting a proposal that the terminal phosphate group of FMN plays a role in decreasing the stability of the hydroquinone complex and in decreasing the redox potential of the semiquinone/hydroquinone couple.
Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage
Authors: DUDHIA Jayesh , DAVIDSON Catherine M. , WELLS Terri M. , VYNIOS Demis H. , HARDINGHAM Timothy E. , BAYLISS Michael T.
Published: August 2015

The content of the C-terminal region of aggrecan was investigated in samples of articular cartilage from individuals ranging in age from newborn to 65 years. This region contains the globular G3 domain which is known to be removed from aggrecan in mature cartilage, probably by proteolytic cleavage, but the age-related changes in its abundance in human cartilage have not been described previously. The analysis was performed by immunosorbant assay using an antiserum (JD5) against recombinant protein expressed from a cDNA clone encoding the terminal 598 amino acid residues of human aggrecan, on crude extracts of cartilage without further purification of aggrecan. The results showed that the content of the C-terminal region decreased with age relative to the G1 domain content (correlation coefficient = 0.463). This represented a 92% fall in the content of this region of the molecule from newborn to 65 years of age. Furthermore, when the G1 content of the cartilage extracts was corrected to only include the G1 attached to aggrecan and to exclude the G1 fragments which accumulate as a by-product of normal aggrecan turnover (free G1), the age-related decrease in the C-terminal region remained very pronounced. Analysis by composite agarose/PAGE showed that the number of subpopulations of aggrecan resolved increased from one in newborn to three in adult cartilage. All of these reacted with an antiserum to the human G1 domain, but only the slowest migrating species reacted with the C-terminal region antiserum (JD5). Similar analysis by SDS/PAGE confirmed the presence of high-molecular-mass (200 kDa) proteins reactive with JD5, but no reactive fragments of lower electrophoretic mobility were detected. In contrast, when probed with the antiserum to the human G1 domain, the immunoblots showed protein species corresponding to the free G1 and G1-G2 fragments, which were present at high concentrations in adult cartilage. The results suggest that the loss of the C-terminal region is not directly part of the process of aggrecan turnover, but it is a slow independent matrix process that occurs more extensively with aging as turnover rates become slower. Young cartilage with the fastest turnover contains least molecules lacking the C-terminal region, whereas in old tissue with slow turnover few molecules retain this region. An increase in the cleavage of this region with age may also contribute to this change. The content of the C-terminal region may thus give a measure of the abundance of newly synthesized aggrecan.
Identification and characterization of a 44 kDa protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization
Authors: GENESTE Olivier , RAFFALLI Françoise , LANG Matti A.
Published: August 2015

Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041–8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA–protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDA RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.
Differential effects of G-protein activators on 5-hydroxytryptamine and platelet-derived growth factor release from streptolysin-O-permeabilized human platelets
Authors: PADFIELD Philip J. , PANESAR Ninder , HENDERSON Patricia , BALDASSARE Joseph J.
Published: August 2015

In this paper we have used streptolysin O (SLO)-permeabilized human platelets to examine the G-protein(s) that control Ca2+-independent secretion from α and dense-core granules. As shown for electropermeabilized platelets, Ca2+ alone stimulated a concentration-dependent increase in 5-hydroxytryptamine (5-HT) (dense-core-granule marker) and platelet-derived growth factor (PDGF) (α-granule marker) release from the SLO-permeabilized cells. The EC50 values for Ca2+-dependent 5-HT and PDGF release were 5 μM and 10 μM respectively. Guanosine 5´-[γ-thio]triphosphate (GTP[S]) (100 μM) stimulated Ca2+-independent release from both α and dense-core granules. In contrast, AlF4- had no effect on Ca2+-independent release from either α or dense-core granules. Neither GTP[S] nor AlF4- appeared to have a significant effect on Ca2+-dependent release from α and dense-core granules. GTP[S] can activate both heterotrimeric and low-molecular-mass G-proteins, whereas AlF4- activates only heterotrimeric G-proteins. Our results, therefore suggest that secretion in the human platelet is regulated by a small G-protein. Both GTP[S]- and Ca2+-dependent secretion were effected by extending the time between permeabilization with SLO and stimulation of secretion. GTP[S]-stimulated secretion from α and dense-core granules decreased rapidly after permeabilization. In contrast, Ca2+-dependent 5-HT and PDGF release ran down at a much lower rate. These observations indicate that GTP[S] and Ca2+ act through parallel pathways to stimulate secretion from SLO-permeabilized platelets.
The role of protein kinase C in carbachol-induced and of cAMP-dependent protein kinase in isoproterenol-induced secretion in primary cultured guinea pig parotid acinar cells
Authors: MÖLLER Karsten , BENZ Diane , PERRIN Dominique , SÖLING Hans-Dieter
Published: August 2015

Stimulation of secretion by muscarinic agonists in guinea pig parotid or pancreatic acini is accompanied by a translocation of protein kinase C (PKC) from the cytosol to the particulate fraction [Machado-De Domenech and Söling (1987) Biochem. J. 242, 749–754] and by a PKC-mediated phosphorylation of the ribosomal protein S6 [Padel and Söling (1985) Eur. J. Biochem. 151, 1–10]. In order to decide whether PKC is directly involved in the secretory process, the effect of down regulation of PKC by phorbol 12-myristate 13-acetate (PMA) was studied in primary cultured guinea pig parotid acinar cells. These cells secrete in response to carbachol and isoproterenol. Only the carbachol response is associated with an increase in cytosolic calcium. Carbachol plus isoproterenol lead to an over-additive stimulation of secretion, an effect which depends completely on the presence of external calcium. Down regulation of PKC by about 90% did not significantly affect carbachol-induced exocytosis, whereas isoproterenol-stimulated secretion was almost doubled. The secretory response to permeable cAMP analogues was also enhanced in PKC-down-regulated acini, indicating a post-receptor effect. The increased response to isoproterenol was also observed in the absence of external calcium. The isoproterenol effect was significantly inhibited by the relatively specific cAMP-dependent protein kinase inhibitor H-89, which had only a minor effect on carbachol-induced exocytosis. Although down regulation of total PKC by up to 90% did not significantly affect the secretory response to carbachol, RO 31-8220, a relatively specific inhibitor of PKC, abolished carbachol-induced secretion in normal as well as in PMA-down-regulated cells. This indicates that a PKC isoform resistant to down regulation by PMA is involved in carbachol- but not in cAMP-mediated secretion.
Intracellular calcium stores and inositol 1,4,5-trisphosphate receptor in rat liver cells
Authors: LIÈVREMONT Jean-Philippe , HILL Anne-Marie , TRAN Dien , COQUIL Jean-François , STELLY Nicole , MAUGER Jean-Pierre
Published: August 2015

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.
Inositol phosphates in the duckweed Spirodela polyrhiza L
Authors: BREARLEY Charles A. , HANKE David E.
Published: August 2015

We have undertaken an analysis of the inositol phosphates of Spirodela polyrhiza at a developmental stage when massive accumulation of InsP6 indicates that a large net synthesis is occurring. We have identified Ins3P, Ins(1,4)P2, Ins(3,4)P2 and possibly Ins(4,6)P2, Ins(3,4,6)P3, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, D- and/or L-Ins(1,2,4,5,6)P5 and InsP6 and revealed the likely presence of a second InsP3 with chromatographic properties similar to Ins(1,4,5)P3. The higher inositol phosphates identified show no obvious direct link to pathways of metabolism of second messengers purported to operate in higher plants, nor do they resemble the immediate products of plant phytase action on InsP6.
Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA
Authors: MICHAEL Anthony J. , FURZE Judith M. , RHODES Michael J. C. , BURTIN Daniel
Published: August 2015

A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription–PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC route to putrescine is present.
Identification of a nuclear-specific cyclophilin which interacts with the proteinase inhibitor eglin c
Authors: WANG Bruce B. , HAYENGA Kirk J. , PAYAN Donald G. , FISHER Joseph M.
Published: August 2015

We have identified a novel human cyclophilin (hCyP-60) which interacts with the proteinase inhibitor eglin c using the yeast two-hybrid system. A cDNA isolated from a Raji B lymphocyte library reveals a domain showing sequence similarity to known cyclophilins flanked by unique N- and C-terminal residues. In addition, hCyP-60 contains a tyrosine residue (Tyr389) instead of a tryptophan residue found in most eukaryotic cyclophilins at a position important for cyclosporin binding. Northern and Western analysis reveal widespread expression with considerable tissue-specific variation. Specifically, the highest levels of mRNA are detected in the thymus, pancreas, testis, and K-562 cell line, while the most protein is detected in the kidney. Immunohistochemistry indicates a nuclear-specific localization both in transfected cells and tissue sections. hCyP-60's specific subcellular localization and conserved amino acid sequence suggest that it may play a specific role in the nucleus.
Insulin and insulin-like growth factor-1 stimulate dephosphorylation of paxillin in parallel with focal adhesion kinase
Authors: KONSTANTOPOULOS Nicky , CLARK Stella
Published: August 2015

Paxillin and focal adhesion kinase (pp125FAK) co-localize in focal adhesions; recently insulin has been shown to stimulate the dephosphorylation of pp125FAK; however, its effect on paxillin is unknown. We show that insulin and IGF-1 can stimulate the dephosphorylation of paxillin in CHOT (overexpress human insulin receptors) and CHO∆CT69 (overexpress insulin receptors lacking C-terminal 69 amino acids) cells. Furthermore, the insulin-receptor C-terminus is not needed for either insulin or IGF-1 to stimulate paxillin or pp125FAK dephosphorylation in the CHO (Chinese-hamster ovary) cell lines used.
Kinetic and spectroscopic properties of the cyanide complexes of ferrous haemoglobins I and IV from trout blood
Authors: ANTONINI Giovanni , BELLELLI Andrea , BRUNORI Maurizio , FALCIONI Giancarlo
Published: August 2015

The cyanide ion is a ligand of ferrous as well as ferric haemoproteins and this study presents a kinetic characterization of the dissociation of its complexes with the two main haemoglobin components from trout blood. Both these haemoglobins bind oxygen co-operatively at neutral or alkaline pH values but one of them is insensitive to pH and allosteric effectors (haemoglobin I, HbI) while the other (haemoglobin IV, HbIV) is strongly sensitive and shows the so-called Root effect (i.e. the incomplete oxygen saturation in air-equilibrated solutions at pH values of < 6.5). Comparison of the kinetics of dissociation of cyanide from ferrous forms of HbI and HbIV reveals that: (i) cyanide dissociates in both cases by a complex reaction, and, at least in the case of HbIV, this may be attributed to functional differences between the α and β subunits; (ii) the reaction is only scarcely co-operative in HbI and not at all so in HbIV; and (iii) the Bohr and Root effects are not manifested in this reaction. The functional heterogeneity of ferrous α and β chains of trout HbI has not been observed for any other ligand; moreover, the observation that co-operativity for cyanide dissociation is expressed by human haemoglobin but not by trout HbIV is surprising.
Probing serpin reactive-loop conformations by proteolytic cleavage
Authors: CHANG Wun-Shaing W. , WARDELL Mark R. , LOMAS David A. , CARRELL Robin W.
Published: August 2015

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the relationship of their reactive-loop conformations to those of circulating forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and anti-thrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop peptides. Proteolysis at the P10–P9, P8–P7 and P7–P6 of anti-trypsin was distorted by binary complex formation. The P1´–P2´ bond in anti-thrombin was more accessible to proteolysis after binary complex formation, whereas cleavage at the P4–P3 bond was variably altered by synthetic peptide insertion. The proteolytic accessibility of the reactive-site P1–P1´ bond of anti-trypsin and anti-thrombin binary complexes was identical with that of the native form and no cleavage was observed in the hinge region (P15–P10) of either protein, whether native or as binary complexes. These results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in the circulation, with the hinge region being partly incorporated into the A β-pleated sheet. This loop can be displaced by peptides and induced to adopt a new conformation similar to the three-turn helix of ovalbumin. Both the native and binary complexed forms of anti-thrombin showed a greatly increased proteolytic sensitivity in the presence of heparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates the approximation of enzyme and inhibitor.