bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein
Authors: CONNOLLY Daniel T , McINTYRE Jonathan , HEUVELMAN Deborah , REMSEN Edward E , McKINNIE Russell E , VU Linh , MELTON Michele , MONSELL Roger , KRUL Elaine S , GLENN Kevin
Published: August 2015

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (Mw) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre·g-1· cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% α-helix, 35% β-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in ‘half-reaction’ experiments.
Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cerevisiae
Authors: IZAWA Shingo , INOUE Yoshiharu , KIMURA Akira
Published: August 2015

Controversy about the importance of catalase in the detoxification of H2O2 in human erythrocytes continues. It has been suggested that catalase has no role in the clearance of H2O2 in erythrocytes. In the present study we investigated the role of catalase in the defence mechanism against oxidative stress using Saccharomyces cerevisiae. S. cerevisiae has two catalases, catalase A and catalase T. We constructed a double mutant (acatalasaemic mutant) unable to produce either catalase A or catalase T, and compared it with wild-type and single-mutant cells. The acatalasaemic mutant cells showed a similar growth rate to wild-type cells under non-oxidative stress conditions, and showed a similar susceptibility to H2O2 stress in the exponential growth phase. The acatalasaemic mutant cells at stationary phase were, however, much more sensitive to H2O2 stress than wild-type and single-mutant cells. Moreover, the ability of acatalasaemic and single-mutant cells to show adaptation to 2 mM H2O2 was distinctly inferior to that of wild-type cells. These results suggest that catalase is not essential for yeast cells under normal conditions, but plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of these cells.
Changes in mRNA levels of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase isoforms in the rat soleus muscle regenerating from notexin-induced necrosis
Authors: ZÁDOR Ernö , MENDLER Luca , HEYEN Mark VER , DUX László , WUYTACK Frank
Published: August 2015

The relative mRNA levels corresponding to the different sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase isoforms (SERCA1a, SERCA1b, SERCA2a, SERCA2b and SERCA3) were measured by reverse transcriptase–PCR in rat soleus muscles regenerating after notexin-induced necrosis. The succession of appearance of the different types of SERCA mRNA species in regenerating muscle largely recapitulates those observed during normal ontogenesis. The mRNA levels of the muscle-specific isoforms SERCA1a and SERCA2a became very low on the first and third days after injection of the snake venom. It was only on the fifth day of regeneration that the mRNA of the neonatal variant of the fast-twitch skeletal SERCA1b isoform began to rise, well before the other SERCA transcripts. At 7 and 10 days, i.e. at a time when the new myofibres normally become re-innervated, the mRNA level of SERCA1a and SERCA2a increased markedly, but the fast-twitch skeletal SERCA1a isoform was still the most prominent. On day 21, in the advanced stage of regeneration, a switch in the relative expression levels of SERCA1a and SERCA2a mRNA was observed and the ratio of both isoforms became similar to that found in the normal soleus muscles. This was followed by a decline in the level of all SERCA mRNA species, so that on day 28 the levels of the sarcoplasmic/endoplasmatic-reticulum Ca2+-pump RNAs was again lower but their ratio remained similar to that of the untreated control soleus.
Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24
Authors: POINOT-CHAZEL Caroline , PORTIER Marielle , BOUABOULA Monsif , VITA Natalio , PECCEU Florence , GULLY Danielle , MONROE John G. , MAFFRAND Jean-Pierre , FUR Gérard LE , CASELLAS Pierre
Published: August 2015

Neurotensin (NT) is a neuropeptide that is important in a variety of biological processes such as signal transduction and cell growth. NT effects are mediated by a single class of cell-surface receptors, known as neurotensin receptors (NTRs), which exhibit structural features of the G-protein-coupled receptors superfamily. We investigated NTR signalling properties with Chinese hamster ovary (CHO) cells stably transformed with human NTR (hNTR). First, we showed that NTR stimulation by NT induced the activation of the mitogen-activated protein kinases (MAPKs) in time- and dose-dependent manners. Both p42 and p44 MAPK isoforms were retarded in gel-shift assays, which was consistent with their activation by phosphorylation. In addition we showed that NT caused a prolonged activation of MAPK as measured by in-gel kinase assay. Secondly, we demonstrated that NT induced the expression of the growth-related gene Krox-24 at the protein level, as assessed by Western-blot analysis, and at the transcriptional level, as demonstrated in CHO cells transfected with hNTR and a reporter gene for Krox-24. Activation of MAPK and induction of Krox-24 were both prevented by the NTR antagonist SR 48692, confirming the specific action on NTR. Furthermore we observed coupling of NTR to a mitogenic pathway and Krox-24 induction in the human adenocarcinoma cell line HT29, which naturally expresses NTRs. Considering coupling pathways between NTR stimulation and MAPK activation, we observed a partial inhibition by pertussis toxin (PTX) and a complete blockade by the protein kinase C (PKC) inhibitor GF 109203X. Taken together, these results suggest that (1) stimulation of NTR activates the MAPK pathway by mechanisms involving dual coupling to both PTX-sensitive and PTX-insensitive G-proteins as well as PKC activation, and (2) these effects are associated with the induction of Krox-24, which might be a target of MAPK effector.
Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis
Authors: MUKHERJEE Tapan , BASU Dhiman , MAHAPATRA Sebabrata , GOFFIN Colette , BEEUMEN Jos van , BASU Joyoti
Published: August 2015

The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR3-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of Mr 52000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin.
Evidence for a novel cytoplasmic tRNA-protein complex containing the KH-multidomain protein vigilin
Authors: KRUSE Charli , GRÜNWELLER Arnold , NOTBOHM Holger , KÜGLER Sebastian , PURSCHKE Werner G. , MÜLLER Peter K.
Published: August 2015

Vigilin, a protein found predominantly in cells and tissues with a high biosynthetic capacity, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Vigilin forms part of a novel ribonucleoprotein complex that also contains additional, as yet uncharacterized, proteins. Experimental evidence suggests that the nucleic acids entrapped in this complex are protected from RNase and belong to the tRNA family. Using either a pool of total human RNA or radioactively labelled tRNA (tRNAAsp**) in rebinding experiments, we could show that tRNA is selectively recaptured by the RNA-depleted vigilin-containing complex.
Construction of a P450c27 fusion enzyme: a useful tool for analysis of vitamin D3 25-hydroxylase activity
Authors: DILWORTH F. Jeffrey , BLACK Stephen M. , GUO Yu-Ding , MILLER Walter L. , JONES Glenville
Published: August 2015

Liver mitochondrial P450c27, encoded by the CYP27 gene, can catalyse the 25-hydroxylation of vitamin D3 and the 27-hydroxylation of sterols. To facilitate the study of this enzyme in cell culture systems, we engineered a fusion protein consisting of P450c27 coupled to its electron-transport accessory proteins, ferredoxin and ferredoxin reductase, and assessed its enzyme activity by measuring the C-25 and C-27 (side-chain) hydroxylation of 1α-hydroxyvitamin D3 (1α-OH-D3). When incubated with 1α-OH-D3, COS-1 cells transfected with a vector expressing the fusion protein produced 1α,25-(OH)2D3 and 1α,27-(OH)2D3 about four times more efficiently than did cells transfected with three individual components of the fusion. When incubated with the natural substrate, vitamin D3, the efficiency of hydroxylation was lower than that for 1α-OH-D3 but still 1.7-fold higher for the fusion protein than for its individual components. The fusion protein was also able to reproduce qualitatively and quantitatively the activity shown by P450c27 in liver cells in situ. The P450c27–ferredoxin reductase–ferredoxin fusion construct represents a valuable tool for establishing the substrate specificity of this liver cytochrome and for evaluating its potential for activating pro-drug analogues of vitamin D.
Internalization and catabolism of radiolabelled antibodies to the MHC class-II invariant chain by B-cell lymphomas
Authors: HANSEN Hans J , ONG Gaik Lin , DIRIL Habibe , VALDEZ Anita , ROCHE Paul A. , GRIFFITHS Gary L , GOLDENBERG David M. , MATTES Jules M.
Published: August 2015

The fate of antibody (Ab) LL1, which reacts with the invariant chain (Ii) subunit of the immature MHC class-II antigen (CD74) after binding to the surface of B-cell lymphomas was investigated. This Ab was internalized and catabolized very rapidly, much faster than other Abs that are considered to be rapidly internalized, such as CD19, CD22 and anti-(transferrin receptor). Such internalization did not depend on Ab cross-linking. The capacity of this uptake process was determined in long-term experiments by increasing the Ab concentration: in 1 day, approx. 8×106 Ab molecules per cell were catabolized. This analysis was facilitated by the use of radiolabels that are trapped within cells after catabolism of the Abs to which they were conjugated. If the Ab is a reliable marker for the Ii antigen, which is likely, we can conclude that Ii directed to the cell surface appears to be sufficient, indeed more than sufficient, to account for the cell content of mature class-II molecules.
Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria
Authors: MILDAZIENE Vida , BANIENE Rasa , NAUCIENE Zita , MARCINKEVICIUTE Ausra , MORKUNIENE Ramune , BORUTAITE Vilmante , KHOLODENKO Boris , BROWN Guy C.
Published: August 2015

Stimulation of mitochondrial respiration by physiological concentrations of Ca2+ was studied to determine which components of oxidative phosphorylation are affected by Ca2+. The kinetic dependence of the respiratory chain, phosphorylation subsystem and proton leak on the mitochondrial membrane potential in isolated rat heart mitochondria respiring on 2-oxoglutarate or succinate was measured at two different concentrations of external free Ca2+. The results show that proton leak is not directly affected by Ca2+, but that both the respiratory and phosphorylation systems can be directly stimulated by Ca2+ depending on conditions. Although Ca2+ directly stimulates the phosphorylation system, this has relatively little effect on respiration rate with 2-oxoglutarate in States 3 and 4 because the subsystem has little control over respiration. However, in intermediate states, the phosphorylation system has greater control and Ca2+ stimulation of this system contributes substantially to the stimulation of respiration and phosphorylation. In the case of succinate oxidation neither the respiratory subsystem nor the phosphorylation system is stimulated by Ca2+.
Two differentially expressed interleukin-11 receptor genes in the mouse genome
Authors: BILINSKI Petra , HALL Mark A. , NEUHAUS Herbert , GISSEL Cornelia , HEATH John K. , GOSSLER Achim
Published: August 2015

Interleukin-11 (IL-11) is a multifunctional cytokine involved in the regulation of cell proliferation and differentiation in a variety of cell types and tissues in vitro and in vivo. The effects of IL-11 were shown to be mediated by the IL-11 receptor (hereafter referred to as IL-11Rα), which is a ligand-binding subunit and provides ligand specificity in a functional multimeric signal-transduction complex with gp130. Here we show that the mouse genome contains a second gene encoding an IL-11-binding protein, referred to as IL-11Rβ. The structure of the IL-11Rβ gene is highly similar to that of IL-11Rα, and IL-11Rβ exhibits 99% sequence identity with IL-11Rα at the amino acid level. IL-11Rβ is co-expressed with IL-11Rα, albeit at lower levels, in embryos and in various adult tissues. IL-11Rβ transcripts are abundant in testis, and, in contrast with IL-11Rα, absent from skeletal muscle. IL-11Rβ expressed in vitro binds IL-11 with high affinity, suggesting that the mouse genome contains a second functional IL-11R.
Comparative studies on the 5-aminolaevulinic acid dehydratases from Pisum sativum, Escherichia coli and Saccharomyces cerevisiae
Authors: M. SENIOR Natalie , BROCKLEHURST Keith , COOPER Jon B. , WOOD Stephen P. , ERSKINE Peter , SHOOLINGIN-JORDAN Peter M. , THOMAS Paul G. , WARREN Martin J.
Published: August 2015

5-Aminolaevulinic acid dehydratase (ALAD) is an essential enzyme in most organisms, catalysing an inaugural step in the tetrapyrrole biosynthetic pathway, the Knorr-type condensation reaction of two molecules of 5-aminolaevulinic acid (ALA) to form the monopyrrole porphobilinogen. ALADs can be conveniently separated into two main groups: those requiring Zn2+ for activity (typified here by the enzymes from Escherichia coli and Saccharomyces cerevisiae, yeast) and those requiring Mg2+ (represented here by the enzyme from Pisum sativum, pea). Here we describe a detailed comparison of these two metal-dependent systems. Kinetically influential ionizations were identified by using pH-dependent kinetics. Groups with pKa values of approx. 7 and 10 (assigned to cysteine and lysine residues) were detected in the free enzyme and enzyme–substrate states of all three enzymes, and a further ionizable group with a pKa of approx. 8.5 (assigned to histidine) was found to be additionally important to the yeast enzyme. The importance of these residues was confirmed by using protein modifying reagents. Shifts in the pKa values of the pea and E. coli enzymes consequent on E–S complex formation suggest a change to a less hydrophobic microenvironment when substrate binds. Studies with inhibitors revealed that the three enzymes exhibit differential susceptibilities and, in the case of succinylacetone, this is reflected in Ki values that vary by three orders of magnitude. In addition, the crystallization of the yeast ALAD is described, raising the possibility of an X-ray-derived three-dimensional structure of this enzyme.
Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport
Authors: SHAROM Frances J. , YU Xiaohong , DiDIODATO Giulio , CHU Joseph W. K.
Published: August 2015

P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport.
Structure-activity relationships for chemical and glutathione S-transferase-catalysed glutathione conjugation reactions of a series of 2-substituted 1-chloro-4-nitrobenzenes
Authors: VAN der AAR Ellen M. , BOUWMAN Tialda , COMMANDEUR Jan N. M. , VERMEULEN Nico P. E.
Published: August 2015

Glutathione S-transferases (GSTs) constitute an important class of phase II (de)toxifying enzymes, catalysing the conjugation of glutathione (GSH) with electrophilic compounds. In the present study, Km, kcat and kcat/Km values for the rat GST 1-1-, 3-3-, 4-4- and 7-7-catalysed conjugation reactions between GSH and a series of 10 different 2-substituted 1-chloro-4-nitrobenzenes, and the second-order rate constants (ks) of the corresponding base-catalysed reactions, were correlated with nine classical physicochemical parameters (electronic, steric and lipophilic) of the substituents and with 16 computer-calculated molecular parameters of the substrates and of the corresponding Meisenheimer complexes with MeS- as a model nucleophile for GS- (charge distributions and several energy values), giving structure–activity relationships. On the basis of an identical dependence of the base-catalysed as well as the GST 1-1- and GST 7-7-catalysed reactions on electronic parameters (among others, Hammett substituent constant σp and charge on p-nitro substituents), and the finding that the corresponding reactions catalysed by GSTs 3-3 and 4-4 depend to a significantly lesser extent on these parameters, it was concluded that the Mu-class GST isoenzymes have a rate-determining transition state in the conjugation reaction between 2-substituted 1-chloro-4-nitrobenzenes and GSH which is different from that of the other two GSTs. Several alternative rate-limiting transition states for GST 3-3 and 4-4 are discussed. Furthermore, based on the obtained structure–activity relationships, it was possible to predict the kcat/Km values of the four GST isoenzymes and the ks of the base-catalysed GSH conjugation of 1-chloro-4-nitrobenzene.
Evidence for a Ca2+ pool associated with secretory granules in rat submandibular acinar cells
Authors: MARTINEZ J. Ricardo , WILLIS Stephanie , PUENTE Sandra , WELLS Julia , HELMKE Ronald , ZHANG Guo H.
Published: August 2015

Intracellular Ca2+ stores in rat submandibular acinar cells were characterized using the Ca2+-sensitive fluorescent indicator fura 2 and the radiotracer 45Ca2+. Acetylcholine induced a rapid Ca2+ release from a store sensitive to inositol 1,4,5-trisphosphate (IP3) and to thapsigargin (TG). After this store was presumably depleted, ionomycin caused a further increase in cytosolic free Ca2+ concentration ([Ca2+]i), suggesting the presence of an IP3-insensitive Ca2+ release from a store that is more extensive and heterogeneous than the IP3-sensitive one and includes a small mitochondrial component. After both of these stores had been discharged, exposure to monensin caused an additional release of Ca2+ from a third store. This store appears to be associated with secretory granules, since Ca2+ release was significantly reduced when degranulation was induced by isoprenaline. This third store appears to be insensitive to IP3, discharges Ca2+ when the pH gradient across the limiting membrane is collapsed with monensin and only in the presence of both ionomycin and monensin. Ca2+ release from this store is not by Na+/Ca2+ exchange, since simply altering [Na+]i did not cause significant Ca2+ release. In permeabilized cells, IP3 and TG released approx. 35% of 45Ca2+, and ionomycin released an additional 57%, whereas monensin only caused a small additional release, suggesting that only IP3- and ionomycin-sensitive stores are loaded with 45Ca2+ under these conditions. The absence of significant isotope uptake into the ionomycin+monensin-sensitive store may result from a low rate of tracer accumulation or from the lack of Ca2+ pumps in the store. The pattern of response was similar in the presence and absence of mitochondrial inhibitors, indicating that the store is not located in mitochondria. In summary, these results suggest that a substantial IP3-insensitive Ca2+ store is present in secretory granules in rat submandibular acinar cells.
Cloning and characterization of a 92 kDa soluble phosphatidylinositol 4-kinase
Authors: NAKAGAWA Tamotsu , GOTO Kaoru , KONDO Hisatake
Published: August 2015

A phosphatidylinositol (PtdIns) 4-kinase cDNA cloned from a rat brain cDNA library encoded a protein of 816 amino acids with a calculated molecular mass of 91654 Da. This molecule contained a lipid-kinase-unique domain and a presumed lipid/protein kinase homology domain that are found in other PtdIns 4-kinases and PtdIns 3-kinases. Furthermore, this kinase molecule had 43.3% shared identity with the presumed catalytic domain of yeast PtdIns 4-kinase, PtdInsK1, and the two molecules had a region of similarity that is not conserved in other lipid kinases. By examining PtdIns kinase activity in transfected COS-7 cells using epitope tag immunoprecipitation as well as conventional methods, the product PtdIns phosphate was identified as phosphatidylinositol 4-phosphate (PtdIns4P), but not phosphatidylinositol 3-phosphate (PtdIns3P). The PtdIns 4-kinase activity was recovered predominantly from the soluble fraction and the activity was markedly enhanced in the presence of Triton X-100 and was relatively insensitive to inhibition by adenosine. In addition, the PtdIns 4-kinase activity was completely inhibited in the presence of 10 µM wortmannin. When examined by epitope tag immunocytochemistry, the immunoreactivity for the PtdIns 4-kinase molecule was dominantly aggregated in a cytoplasmic region juxtaposed to the nuclei and was faintly but widely dispersed in the cytoplasm. By in situ hybridization analysis, the mRNA for PtdIns 4-kinase was expressed ubiquitously and was detected in most neurons throughout the grey matter of the brain, with higher expression intensity found in fetal than in adult brain.
Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles
Authors: WESTERMANN Peter , KNOBLICH Maria , MAIER Olaf , LINDSCHAU Carsten , HALLER Hermann
Published: August 2015

Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vitro of 35SO4-labelled HSPGs into transport vesicles was investigated. Formation of transport vesicles at the trans-Golgi network was stimulated by PMA and inhibited by calphostin C or Ro 31-8220 by using a post-nuclear supernatant. Treatment of either isolated Golgi-enriched membranes or cytosolic proteins with calphostin C provided evidence that membrane-bound PKC forms strongly supported vesicle formation, whereas cytosolic PKC forms showed a marginal effect. The PKC isoforms PKC-α and PKC-ζ were attached to highly purified Golgi membranes, as shown by Western blotting. Both isoforms were localized by confocal immunofluorescence microscopy in the Golgi area of HepG2 cells. Immunoelectron microscopy of ultrathin cryosections of HepG2 cells showed that PKC-ζ predominantly attaches to the trans-Golgi region, whereas PKC-α binds to the cis- and trans-Golgi area.
Histamine reduces ZO-1 tight-junction protein expression in cultured retinal microvascular endothelial cells
Authors: GARDNER Thomas W. , LESHER Treena , KHIN Sonny , VU Cuong , BARBER Alistair J. , BRENNAN William A.
Published: August 2015

We examined ZO-1 protein content in cultured retinal vascular endothelial cells to test the hypothesis that histamine alters tight-junction-protein expression. Histamine (10-9 –10-4 M) causes a reversible concentration-dependent reduction of ZO-1 protein content, mediated by both H1 and H2 receptors. Histamine reduces ZO-1 expression within the time associated with increased paracellular permeability. Tight-junction-protein alterations may be a novel explanation for the mechanism by which vasoactive agents increase microvascular permeability.
Insulin action in cultured human myoblasts: contribution of different signalling pathways to regulation of glycogen synthesis
Authors: HUREL Steven J. , ROCHFORD Justin J. , BORTHWICK Andrew C. , WELLS Anne M. , VANDENHEEDE Jackie R. , TURNBULL Douglass M. , YEAMAN Stephen J.
Published: August 2015

A key metabolic action of insulin is the stimulation of non-oxidative glucose utilization in skeletal muscle, by increasing both glucose uptake and glycogen synthesis. The molecular mechanism underlying this process has been investigated using a variety of experimental systems. We report here the use of cultured human myoblasts to study insulin control of glycogen synthesis in humans. In these cells insulin stimulates glycogen synthesis approx. 2.2-fold, associated with a similar activation of glycogen synthase (GS) which occurs within 5–10 min of the addition of insulin. Insulin also causes inactivation of glycogen synthase kinase-3 (GSK-3) and activation of protein kinase B, both processes being sufficiently rapid to account for the effects of insulin on GS. Activation by insulin of the protein kinases p70s6K, p90s6K and extracellular signal-regulated kinase 2 (ERK2) is observed, but is significantly slower than the activation of GS. Selective inhibitors of the p70s6K pathway (rapamycin), the ERK2/p90s6K pathway (PD98059) and phosphatidylinositol 3-kinase (wortmannin) have been used to probe the contribution of these components to insulin signalling in human muscle. Wortmannin blocks activation of both glycogen synthesis and GS and inactivation of GSK-3. PD98059 is without effect on these events, while rapamycin is without effect on inactivation of GSK-3 but partially blocks activation of glycogen synthesis and GS. Taken together, these findings suggest that protein kinase B is responsible for the inactivation of GSK-3, but that an additional rapamycin-sensitive mechanism may contribute to the activation of GS and stimulation of glycogen synthesis.
Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism
Authors: PETRYNIAK Magdalena A. , WURTMAN Richard J. , SLACK Barbara E.
Published: August 2015

Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337–8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism.
Adipocyte differentiation-related protein is secreted into milk as a constituent of milk lipid globule membrane
Authors: HEID Hans W. , SCHNÖLZER Martina , KEENAN Thomas W.
Published: August 2015

Milk lipid globules from humans, cows and rats contained a protein identified as adipocyte differentiation-related protein (ADRP) associated with the globule surface membrane material. This protein, previously believed to be specific to adipocytes, was a major constituent of the globule surface and was present in a detergent-insoluble complex that contained stoichiometric amounts of butyrophilin and xanthine oxidase. Identification of ADRP was by sequence similarity of tryptic peptides from cow and human proteins with the sequence inferred from the cDNA for mouse ADRP. The putative ADRP of lipid globules from cow, human and rat milk was recognized specifically by antisera raised against a peptide synthesized to duplicate the N-terminal 26 residues of the mouse protein. In homogenates of lactating mammary gland, ADRP was found only in endoplasmic reticulum and in lipid droplet fractions. ADRP was modified, apparently post-translationally, and one modification apparently was acylation, primarily with C14, C16 and C18 fatty acids. Two isoelectric variants of ADRP were present in cow globule membrane material. In vitro, ADRP served as a substrate for protein kinases associated with milk lipid globule membrane, but this protein did not seem to become phosphorylated intracellularly.
Effect of genotype on the levels of surfactant protein A mRNA and on the SP-A2 splice variants in adult humans
Authors: KARINCH Anne M. , deMELLO Daphne E. , FLOROS Joanna
Published: August 2015

Human pulmonary surfactant protein A (SP-A) is encoded by two genes, SP-A1 and SP-A2, that exhibit coding sequence (allelic) and 5´ splicing variability. In this report we determine the effect of the genetic variability within the SP-A1 and SP-A2 genes on the level of SP-A mRNAs and on the SP-A2 splicing variants in different individuals. We analysed mRNA specimens from 23 unrelated adults using genotype analysis, Northern analysis and primer extension, and made the following observations. (1) The level of SP-A mRNA varies among individuals (coefficient of variation = 0.49). One SP-A genotype (6A26A21A01A0) appears to be associated with a low to moderate level of SP-A mRNA. (2) The SP-A1/SP-A2 mRNA ratio varies among individuals, from 0.94 (lowest) to 6.80 (highest) within the study population. One genotype appears to be associated with a moderate to high SP-A1/SP-A2 mRNA ratio and another with a low to moderate ratio. (3) There is no correlation between the level of SP-A mRNA and the SP-A1/SP-A2 mRNA ratio. (4) Variability in the ratio of the major SP-A2 splice variants among individuals results from nucleotide differences in the splice-recognition sequence of specific SP-A2 alleles. The SP-A mRNA levels, the SP-A1/SP-A2 mRNA ratio, and the ratio of the major SP-A2 splice variants have a genetic basis in that they vary depending upon the specific SP-A alleles present.
Interaction between ATP, oleandomycin and the OleB ATP-binding cassette transporter of Streptomyces antibioticus involved in oleandomycin secretion
Authors: BUCHE André , MÉNDEZ Carmen , SALAS José A.
Published: August 2015

The OleB protein of Streptomyces antibioticus, oleandomycin (OM) producer, constitutes an ATP-binding cassette transporter containing two nucleotide-binding domains and is involved in OM resistance and its secretion in this producer strain. We have characterized some properties of the first nucleotide-binding domain of OleB using an overexpressed fusion protein (MBP–OleB´) between a maltose-binding protein (MBP) and the first half of OleB (OleB´). Extrinsic fluorescence of the base-modified fluorescent nucleotide analogue 1,N6-ethenoadenosine 5´-triphosphate (εATP) and 2´(3´)-o-(2,4,6-trinitrophenyl)adenosine-5´-triphosphate was determined in the presence of MBP and the fusion protein MBP–OleB´, and it was found that εATP binds to MBP–OleB´ with a stoichiometry of 0.9. Measurements of the intrinsic fluorescence of the MBP–OleB´ fusion protein indicated that ATP induces a decrease in the accessibility of the MBP–OleB´ tryptophans to acrylamide, an indication of a folding effect. This conclusion was confirmed by the fact that ATP also induces considerable stabilization against guanidine chloride denaturation of MBP–OleB´. Two effects were found to be associated with the presence of Mg2+ ions: (1) an increase in the quenching of MBP–OleB´ intrinsic fluorescence by ATP; and (2) an increase in the accessibility of MBP–OleB´ tryptophans to acrylamide. Significant changes in the intrinsic fluorescence of the fusion protein were also observed in the presence of OM, demostrating the existence of interaction between the transporter and the antibiotic in the absence of any hydrophobic membrane component.
Marked increases in concentrations of apolipoprotein in the cerebrospinal fluid of poliovirus-infected macaques: relations between apolipoprotein concentrations and severity of brain injury
Authors: SAITO Kuniaki , SEISHIMA Mitsuru , HEYES Melvyn P. , SONG Hua , FUJIGAKI Suwako , MAEDA Satoshi , VICKERS James H. , NOMA Akio
Published: August 2015

Apolipoproteins in cerebrospinal fluid (CSF) might have important functional roles in the pathophysiology of brain and lipid metabolism in the vascular component. The present study examined apolipoprotein A-I (apo-A-I) and apolipoprotein E (apo-E) levels in CSF and serum from poliovirus-infected macaques. Poliovirus-infected macaques developed motor deficits and were classified into three groups: (1) muscle weakness in one or both legs; (2) partial paralysis in one or both legs; (3) complete paralysis in one or both legs. No motor deficits were evident in the control or sham-treated macaques. Apo-A-I concentrations in CSF were markedly elevated in poliovirus-infected macaques with weakness, partial or complete paralysis, in comparison with either control or sham-treated animals, and were proportional to the severity of motor impairment. Apo-E concentrations in CSF were also significantly elevated in poliovirus-infected macaques with complete paralysis. The magnitude of increase in CSF apo-A-I or apo-E concentrations was also closely associated with the degree of histologic neurological damage and inflammation (lesion scores). However, no changes in serum apo-A-I and apo-E concentrations were observed in the poliovirus-infected macaques compared with control macaques. Furthermore there were no significant correlations apo-A-I or apo-E concentrations between serum and CSF. We hypothesize that the elevation of apo-A-I and apo-E concentrations after poliovirus infection is caused by immune stimulation within the central nervous system (CNS). Measures of CSF apo-A-I and apo-E levels might serve as a useful marker for the severity and/or the range of CNS injury.
A novel method for the study of autophagy: destruction of hepatocytic lysosomes, but not autophagosomes, by the photosensitizing porphyrin tetra(4-sulphonatophenyl)porphine
Authors: STRØMHAUG Per Eivind , BERG Trond Olav , BERG Kristian , SEGLEN Per O.
Published: August 2015

A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS4), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-β-d-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS4 caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 °C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS4 also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed 125I-tyramine-cellobiose-asialo-orosomucoid and TPPS4 from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic–endocytic–lysosomal interactions.
Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis
Authors: WINTER Elke , BRUMMEL Monika , SCHUCH Ricardo , SPENER Friedrich
Published: August 2015

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalents NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP.
Decreased carbonic anhydrase III levels in the liver of the mouse mutant ‘toxic milk’ (tx) due to copper accumulation
Authors: GRIMES Andrew , PAYNTER Jennifer , WALKER Ian D. , BHAVE Mrinal , MERCER Julian F. B.
Published: August 2015

The mouse mutant ‘toxic milk‘ (tx) is characterized by marked hepatic accumulation of copper, similar to that found in patients with the genetic disorder of copper transport, Wilson disease. In addition, lactating tx females produce copper-deficient milk. To characterize further the biochemical basis of this defect, Western blots of tissue extracts from normal and tx mice were probed with various heavy-metal radioisotopes (63Ni, 65Zn and 64Cu). A 30 kDa Ni/Zn-binding polypeptide was found to be markedly decreased in the livers of the tx mice. This protein was isolated from normal adult mice using a procedure based on Ni-chelation chromatography. The amino acid sequences of two CNBr peptides were identical with portions of the mouse skeletal muscle carbonic anhydrase III (CAIII) sequence. Two other peptides sequenced had closely related sequences to that of CAIII, but with two differences in 45 amino acids. These two peptides may be derived from a novel CAIII isoform, which we term CAIIIB to distinguish it from the published form, CAIIIA. We isolated a cDNA clone corresponding to CAIIIA and used this to show that CAIIIA mRNA was also decreased in the mutant liver, but not in muscle. Copper loading of normal mice also decreased hepatic CAIIIA mRNA, suggesting that the decrease in CAIII mRNA in the tx mouse liver is a secondary consequence of the high copper levels in the liver.
Regulation of the thyroid NADPH-dependent H2O2 generator by Ca2+: studies with phenylarsine oxide in thyroid plasma membrane
Authors: GORIN Yves , LESENEY Anne Marie , OHAYON Renée , DUPUY Corinne , POMMIER Jacques , VIRION Alain , DÈME Danielle
Published: August 2015

Pig thyroid plasma membranes contain a Ca2+-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca2+-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca2+-desensitized by α-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the Km for NADPH determined with Ca2+ (18 ƁM, identical with that of the native enzyme) was approx. one-third of the Km measured without Ca2+, showing the direct action of Ca2+ on the PAOŐenzyme complex. PAO had the same effects, partial inactivation and Ca2+ desensitization, on the NADPH:ferricyanide oxidoreductase activity of the NADPH oxidase, suggesting that PAO acts on the flavodehydrogenase entity of the enzyme. Both partial inactivation and Ca2+ desensitization were completely and specifically reversed by 2,3-dimercaptopropanol, partly reversed by dithiothreitol and not reversed by 2-mercaptoethanol, indicating that PAO binds to vicinal thiol groups. These results suggest that thiol groups are involved in the control of thyroid NADPH oxidase by Ca2+; PAO bound to vicinal thiols might alter the structure of the enzyme so that electron transfer occurs without Ca2+ but more slowly.
Interleukin 1-β-induced protein kinase C-ζ activation is mimicked by exogenous phospholipase D
Authors: LIMATOLA Cristina , BARABINO Benedetta , NISTA Anna , SANTONI Angela
Published: August 2015

Interleukin 1-α (IL1-α) is a pleiotropic cytokine that stimulates a number of signal transduction pathways in cells, leading to different cellular responses. In this study we investigated the signal transduction pathways activated by IL1-α in two different human cell lines: RD/TE671, a rhabdomyosarcoma, and EJ, a bladder-derived carcinoma. We showed that this cytokine induced the activation of protein kinase C-ζ (PKC-ζ) and the accumulation of a putative physiological PKC-ζ activator, phosphatidic acid [Limatola, Schaap, Moolenaar and van Blitterswijk (1994) Biochem. J. 304, 1001Ő1008]. Exogenously supplied phospholipase D, which generated cellular phosphatidic acid, was able to mimic the cytokine effect, supporting the hypothesis that this lipid second messenger might contribute to cytokine-induced PKC-ζ activation. In addition, we show that IL1-α stimulation of BOSC23 cells, transiently overexpressing PKC-ζ, induced an increase in PKC-ζ autophosphorylation. These results give the first direct evidence that IL1-α can activate this atypical PKC isoform and suggest that this enzyme might be involved in mediating some of the biological effects induced by IL1-α.
Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis
Authors: WOLOSKER Herman , ROCHA Joao B. T. , ENGELENDER Simone , PANIZZUTTI Rogerio , MIRANDA Joari De , MEIS Leopoldo de
Published: August 2015

The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effects were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.