bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoaffinity purification of avermectin-binding proteins from the free-living nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster
Authors: Rohrer S P , Jacobson E B , Hayes E C , Birzin E T , Schaeffer J M
Published: August 2015

Avermectin-binding proteins from the free-living nematode worm Caenorhabditis elegans and from the fruitfly Drosophila melanogaster were purified to homogeneity via a three-step procedure. The binding proteins were covalently labelled using a radioactive photoaffinity probe and then partially purified on a Sephacryl S-300 gel-filtration column. The radiolabelled binding proteins were then purified by immunoaffinity chromatography using a monoclonal antibody to avermectin covalently attached to Protein A-Sepharose beads. Three affinity-labelled Drosophila proteins with molecular masses between 45 and 50 kDa were isolated in this way and then separated from each other by electroelution. This three-step protocol provides a rapid technique for receptor purification which may be of use in the purification of other binding proteins.
Promoter elements in the transcriptional activation of the human stromelysin-1 gene by the inflammatory cytokine, interleukin 1
Authors: Quinones S , Buttice G , Kurkinen M
Published: August 2015

Stromelysin-1, a tissue-remodelling metalloproteinase synthesized by fibroblasts, has proteolytic activity against a variety of extracellular matrix components. Stromelysin-1 gene transcription is induced by the inflammatory cytokine interleukin (IL)-1. In fibroblasts transiently transfected with constructs containing 5′-deletion mutants of the human stromelysin-1 gene promoter, IL-1-induced transcriptional activity was abolished with the removal of region -102 to -54. This region includes an AP-1 binding site at positions -70 to -64. The AP-1 site alone increased the basal activity of and conferred minimal IL-1 inducibility onto the heterologous gene promoter of thymidine kinase. Interestingly, although the removal of the AP-1 site from the native promoter (-1303 to +4) affected the absolute levels of IL-1-induced and basal promoter activity, it did not alter their ratio, indicating the involvement of regions outside the AP-1 site in the IL-1 response. Of the stromelysin-1 5′ flanking sequence examined, only the region -274 to -54 could confer IL-1 inducibility to a heterologous promoter independently of the AP-1 site. This region also bound specific nuclear factors. Further analysis revealed that the region composed of -86 to -71 and -63 to -54 could independently respond to IL-1 and bind protein of whole cell extracts. Protein binding to this region and to the AP-1 site was modestly induced by IL-1 treatment. From these results we conclude that, in fibroblasts, the AP-1 site (-70 to -64) is not necessary for the IL-1 response; however, it probably interacts through protein associations with the responsive region immediately surrounding it in the absolute transcriptional activation of the human stromelysin-1 gene by IL-1.
The effect of elevated plasma phenylalanine levels on protein synthesis rates in adult rat brain
Authors: Dunlop D S , Yang X R , Lajtha A
Published: August 2015

Increasing the plasma phenylalanine concentration to levels as high as 0.560-0.870 mM (over ten times normal levels) had no detectable effect on the rate of brain protein synthesis in adult rats. The average rates for 7-week-old rats were: valine, 0.58 +/- 0.05%/h, phenylalanine, 0.59 +/- 0.06%/h, and tyrosine, 0.60 +/- 0.09%/h, or 0.59 +/- 0.06%/h overall. Synthesis rates calculated on the basis of the specific activity of the tRNA-bound amino acid were slightly lower (4% lower for phenylalanine) than those based on the brain free amino acid pool. Similarly, the specific activities of valine and phenylalanine in microdialysis fluid from striatum were practically the same as those in the brain free amino acid pool. Thus the specific activities of the valine and phenylalanine brain free pools are good measures of the precursor specific activity for protein synthesis. In any event, synthesis rates, whether based on the specific activities of the amino acids in the brain free pool or those bound to tRNA, were unaffected by elevated levels of plasma phenylalanine. Brain protein synthesis rates measured after the administration of quite large doses of phenylalanine (> 1.5 mumol/g) or valine (15 mumol/g) were in agreement (0.62 +/- 0.01 and 0.65 +/- 0.01%/h respectively) with the rates determined with infusions of trace amounts of amino acids. Thus the technique of stabilizing precursor-specific activity, and pushing values in the brain close to those of the plasma, by the administration of large quantities of precursor, appears to be valid.
Biosynthesis and polarized distribution of neutral endopeptidase in primary cultures of kidney proximal tubule cells
Authors: Jalal F , Dehbi M , Berteloot A , Crine P
Published: August 2015

When cultured in defined medium, kidney proximal convoluted tubule (PCT) cells form a homogeneous population and retain a number of differentiated functions. To characterize this cell system further as a functional model of epithelial polarity, we investigated the biogenic pathway of neutral endopeptidase (NEP), one of the most abundant microvillar membrane proteins in intestinal and kidney cells. We showed that, in contrast with some tumoral cell lines, RNA extracted from PCT cells shows the presence of a single mRNA species encoding NEP. Pulse-chase studies followed by selective immunoprecipitation of NEP molecules present either at the cell surface or in intracellular cell compartments showed that newly synthesized NEP molecules reached the cell surface as early as 30 min after the beginning of the chase with maximum cell surface expression at 60 min. When grown on semipermeable supports, PCT cells were found to target NEP exclusively to the apical plasma membrane. Similar results have been described using MDCK cells to study targeting of recombinant NEP. Thus primary cultures of PCT cells represent a new model with which to investigate the biogenic pathway of endogenous proteins in native epithelial cells.
Down-regulation of the c-myc proto-oncogene in inhibition of vascular smooth-muscle cell proliferation: a signal for growth arrest?
Authors: Bennett M R , Littlewood T D , Hancock D C , Evan G I , Newby A C
Published: August 2015

Vascular smooth muscle (VSM) cell proliferation contributes to the pathogenesis of atherosclerosis, restenosis after angioplasty and vein graft disease. The regulation of genes involved in VSM cell proliferation, particularly by naturally occurring inhibitors, is therefore of some importance. We have investigated the role of the c-myc proto-oncogene in growth arrest of exponentially proliferating rat VSM cells, following mitogen withdrawal, treatment with heparin (50 micrograms/ml), interferon-gamma (IFN-gamma) (100 i.u./ml), or the cyclic nucleotide analogues, 8-bromo-adenosine-3′5′-cyclic monophosphate (8-Br-cAMP; 0.1 mM) and 8-bromoguanosine-3′5′-cyclic monophosphate (8-Br-cGMP; 0.1 mM). Growth arrest was accompanied by down-regulation of c-Myc protein and mRNA following treatment with all inhibitors. Serum withdrawal or IFN-gamma treatment suppressed c-myc expression by more than 50% within 2 h, and this occurred throughout the cell cycle. Platelet-derived growth factor, epidermal growth factor and basic fibroblast growth factor all contributed independently to the maintenance of c-myc expression. Heparin, 8-Br-cAMP or 8-Br-cGMP also suppressed c-myc, but this occurred later, after 24-48 h, and was also observed following arrest by metabolic block. We conclude that c-myc expression is linked to VSM cell growth arrest in response to endogenous regulators and metabolic block. Down-regulation of c-myc expression may thus be an essential part of the arrest programme in VSM cells induced by many pharmacological agents.
Inositol trisphosphate metabolism in Saccharomyces cerevisiae: identification, purification and properties of inositol 1,4,5-trisphosphate 6-kinase
Authors: Estevez F , Pulford D , Stark M J , Carter A N , Downes C P
Published: August 2015

Ins(1,4,5)P3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity approximately 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of approximately 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P3. The Km for ATP was 2.1 mM, and that for Ins(1,4,5)P3 was 7.1 microM. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P3 into an InsP5, and the partially purified preparation contained another activity that converted the InsP4 product into an InsP5. The InsP4 product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P4 [L-Ins(1,4,5,6)P4] standard, identifying it as D-Ins(1,4,5,6)P4. The yeast enzyme is thus an Ins(1,4,5)P3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP5 and InsP6 in S. cerevisiae.
Is respiratory activity in the brain mitochondria responsive to thyroid hormone action?: a critical re-evaluation
Authors: Katyare S S , Bangur C S , Howland J L
Published: August 2015

The effects of in vivo treatment with graded doses (0.5-1.5 micrograms/g body weight) of thyroid hormones, tri-iodothyronine (T3) and thyroxine (T4), for 4 consecutive days to euthyroid rats on the respiratory activity of isolated brain mitochondria were examined. T4 stimulated coupled State-3 respiration with glutamate, pyruvate + malate, ascorbate + tetramethyl-p-phenylenediamine and succinate, in a dose-dependent manner; T3 was effective only at the highest (1.5 micrograms) dose employed. T4 was more effective than T3 in stimulating respiratory activity. State-4 respiratory rates were in general not influenced except in the case of the ascorbate + tetramethyl-p-phenylenediamine system. Primary dehydrogenase activities, i.e. glutamate dehydrogenase, malate dehydrogenase and succinate dehydrogenase, were stimulated about 2-fold; interestingly mitochondrial but not cytosolic malate dehydrogenase activity was influenced under these conditions. The hormone treatments did not greatly influence the mitochondrial cytochrome content. The results therefore suggest that thyroid hormone treatment not only stimulates primary dehydrogenase activities but may also directly influence the process of mitochondrial electron transfer.
Divergent sequences in the 5′ region of cDNA suggest alternative splicing as a mechanism for the generation of carnitine acetyltransferases with different subcellular localizations
Authors: Corti O , DiDonato S , Finocchiaro G
Published: August 2015

We identified two partially overlapping cDNAs containing divergent 5′ sequences of human carnitine acetyltransferase (CAT). cDNA lambda SM-1400 extends the sequence of peroxisomal CAT, whereas cDNA lambda SM-1200 creates a new open reading frame encoding a putative mitochondrial leader peptide. An intron is located where sequences diverge, suggesting that mitochondrial, peroxisomal and possibly endoplasmic reticulum CAT mRNAs derive from alternative splicing of the CAT gene.
Effects of the S-adenosylmethionine decarboxylase inhibitor, 5′-{[(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine, on cell growth and polyamine metabolism and transport in Chinese hamster ovary cell cultures
Authors: Byers T L , Wechter R S , Hu R H , Pegg A E
Published: August 2015

The regulation of polyamine transport and the roles of polyamine transport and synthesis in cell growth were investigated using cultured Chinese hamster ovary (CHO) cells and CHOMG cells which are mutants lacking polyamine-transport activity. Metabolically stable methylated polyamine analogues were used to measure polyamine accumulation, and the irreversible S-adenosyl-L-methionine decarboxylase inhibitor, 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine (AbeAdo), was used to inhibit synthesis. Exposure to AbeAdo lead to a dose-dependent decrease in growth for both cell lines, although CHOMG cells were more sensitive. Intracellular putrescine levels were greatly increased in AbeAdo-treated CHO cells and to a lesser extent in CHOMG cells, whereas intracellular spermidine and spermine levels were substantially reduced in both. Treatment with AbeAdo increased putrescine content in the culture medium to a much greater extent in CHOMG cultures indicating that a portion of the excess putrescine synthesized in response to AbeAdo treatment is excreted, but that CHO cells salvage this putrescine whereas it is lost to CHOMG cells which cannot take up polyamines. AbeAdo treatment increased polyamine transport into CHO cells despite high intracellular putrescine, suggesting that spermidine and/or spermine, and not putrescine, are the major factors regulating transport activity. The accumulation of either 1-methylspermidine or 1,12-dimethylspermine was significantly increased by AbeAdo treatment. Accumulation was increased even further when protein synthesis was blocked by cycloheximide, indicating that a short-lived protein is involved in the regulation of polyamine uptake. In the presence of cycloheximide and AbeAdo or alpha-difluoromethylornithine, methylated polyamine derivatives accumulated to very high levels leading to cell death. These results show that the polyamine-transport system plays an important role in retaining intracellular polyamines and that down-regulation of the transport system in response to increased intracellular polyamine content is necessary to prevent accumulation of toxic levels of polyamines.
Thaliporphine selectively inhibits expression of the inducible, but not the constitutive, nitric oxide synthase
Authors: Yu S M
Published: August 2015

Formation of nitrites/nitrates caused by lipopolysaccharide (LPS) in J774.2 macrophages was inhibited by thaliporphine, an aporphine derivative isolated from the plant Neolitsea konishii K. This inhibition of nitrite synthesis in LPS-stimulated macrophages by thaliporphine was similar to that by cycloheximide, NG-methyl-L-arginine (MeArg) and dexamethasone. Thaliporphine, but not MeArg, inhibited expression of inducible NO synthase without directly affecting enzyme activity. However, thaliporphine did not inhibit nitrite production by NO synthase that had already been induced by prior exposure to LPS for which any possible further induction was inhibited by cycloheximide. In endothelial cells, nitrite formation induced by bradykinin (in the presence of 0.2 mM Ca2+) was inhibited by MeArg. However, incubation of endothelial cells with dexamethasone, cycloheximide and thaliporphine did not affect this Ca(2+)-dependent nitrite production. Thaliporphine (0.1-100 microM) dose-dependently inhibited nitrite accumulation in macrophages stimulated by interleukin-1 beta (IL-1 beta) whereas nitrite formation induced by tumour necrosis factor alpha was not inhibited. LPS-stimulated IL-1 beta synthesis in macrophages was significantly inhibited by thaliporphine, but thaliporphine had only minimal effect on LPS-stimulated IL-1 beta synthesis in endothelial cells. These results demonstrate that thaliporphine inhibits LPS induction of NO synthase expression, and that the mechanism of action of thaliporphine is via inhibition of LPS-stimulated IL-1 beta synthesis in macrophages. In anaesthetized rats subjected to LPS, pretreatment with thaliporphine partially restored the fall in mean arterial pressure and the vascular hyporeactivity to noradrenaline 3 h after LPS injection. In conclusion, thaliporphine selectively inhibited expression of inducible NO synthase, and may thus hold potential for the treatment of endotoxaemia.
Switching from α1- to β-subtypes in adrenergic response during primary culture of adult-rat hepatocytes as affected by the cell-to-cell interaction through plasma membranes
Authors: Kajiyama Y , Ui M
Published: August 2015

The alpha 1-adrenergic response was predominant over the beta-adrenergic one in adult rat hepatocytes, when the responses were measured as the agonist-induced generations of Ins(1,4,5)P3 and cyclic AMP, respectively. During primary culture of the adult rat hepatocytes, the beta-adrenergic response developed rapidly, whereas the alpha 1-response decreased gradually. Such receptor-subtype switching did not occur unless the cells were cultured under conditions favourable for cell growth, i.e. at low cell density (10(4) cells/cm2). The switching was prevented progressively as the cell culture density was increased up to 20-fold or the low-density culture was achieved by addition of increasing amounts of liver plasma membranes. The gradual decrease in alpha 1-response was accounted for by a concurrent decrease in the receptor site density, whereas rapid development of the beta-response definitely preceded the increase in beta-ligand binding sites during the culture. This rapid development of the beta-response reflected enhanced coupling of the receptor to G-protein during the early stage of culture, as evidenced by the progressively developed ability of GTP to lower the affinity of beta-agonist binding to membranes prepared from these short-time-cultured hepatocytes.
Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators
Authors: Lau L F , Pumiglia K , Côté Y P , Feinstein M B
Published: August 2015

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5′-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5′-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors.
The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca2+-ATPase
Authors: Hughes G , East J M , Lee A G
Published: August 2015

The peptide MEKVQYLTRSAIRRASTIEMPQQAR-Cys corresponding to residues 1-25 of phospholamban was found to inhibit the ATPase activity of skeletal muscle Ca(2+)-ATPase, but to have no effect on the Ca(2+)-dependence of its activity. The peptide was found to decrease the rate of the Ca2+ transport step (E1PCa2-->E2P) by a factor of 2.4. The rate of this same step was decreased by poly(L-Arg) by a factor of 2.2. The peptide shifted the E2-E1 equilibrium of the ATPase towards E1 by a factor of 4 due to stronger binding to the E1 than to the E2 conformation of the ATPase; dissociation constants for binding to E1 and E2 were estimated as 3 and 10 microM respectively. The peptide had no effect on the level of phosphorylation by Pi in the absence of Ca2+ or on the rate of phosphorylation by ATP in the presence of Ca2+.
Kinetics of creatine uptake in the perfused mouse liver: a 31P-n.m.r. study of transgenic mice expressing creatine kinase (CKBB) in the liver
Authors: Masson S , Quistorff B
Published: August 2015

Transport of creatine in the mouse liver has been investigated in vivo and in the perfused organ. Experiments were carried out with transgenic mice expressing creatine kinase in the liver (brain isoenzyme CKBB; EC 7.2.3.2.) [Koretsky, Brosnan, Chen, Chen and Van Dyke (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3112-3116] and in the corresponding control mice. The animals were fed a regular chow with or without the addition of 10% creatine (w/w) for 5 days. The kinetics of creatine uptake was measured in the perfused liver by 31P-n.m.r. spectroscopy and biochemical analysis following infusion of creatine at concentrations ranging over 0-15 mM and at an extracellular pH of either 7.40 or 6.40. The results suggest that creatine is actively transported by a pH-dependent mechanism obeying a saturable Michaelis-Menten type of kinetics (Km = 0.80 +/- 0.18 and 5.12 +/- 2.40 mM; Vmax. = 0.57 +/- 0.04 and 1.72 +/- 0.32 mumol.g of liver-1.min-1 at pH 7.40 and 6.40 respectively). Creatine export was evaluated in the perfused liver preloaded with creatine and the results show that less than 2.5 and 5% of the total creatine pool is exported to the perfusate during 80 min of perfusion at pH 7.40 and 6.40 respectively. Taken together, these results seem to explain the observation that creatine accumulates in the mouse liver only when blood creatine is raised by creatine feeding.
N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation
Authors: Galbiati F , Guzzi F , Magee A I , Milligan G , Parenti M
Published: August 2015

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.
Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum
Authors: Bush K T , Hendrickson B A , Nigam S K
Published: August 2015

In order to determine whether the endoplasmic reticulum (ER) luminal FK506-binding protein, FKBP13, shares properties of ER molecular chaperones, MDCK cells were treated with either tunicamycin or Ca2+ ionophores. By Northern-blot analysis, tunicamycin resulted in a 2-fold rise in FKBP13 mRNA, whereas ionophores (A23187 and ionomycin) caused a more impressive rise in FKBP13 mRNA (up to 5-fold with ionomycin). Actinomycin D chase experiments in ionomycin-treated cells revealed no change in the half-life of FKBP13 mRNA, indicating that the increase in FKBP13 mRNA observed was not due to greater message stability. Moreover, sequencing of the 5′ flanking region of the gene for murine FKBP13 revealed significant similarity to similar regions in human BiP (immunoglobulin-binding protein) and the human glucose-regulated protein grp94, including a 37 bp sequence in FKBP13 with approximately 50% identity with the unfolded protein response element of the BiP gene. Together, these data suggest a role for FKBP13 in ER protein folding.
Human cytochrome b561: a revised hypothesis for conformation in membranes which reconciles sequence and functional information
Authors: Srivastava M , Gibson K R , Pollard H B , Fleming P J
Published: August 2015

Cytochrome b561 is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles. In this report, we describe the cloning and properties of a full-length cDNA encoding human neuroendocrine cytochrome b561 from a human caudate cDNA library and a human peripheral blood genomic library. The human cDNA contains two major transcription start sites and only one translation start site that codes for an apocytochrome b561, which is 22 amino acid residues smaller than the previously deduced amino acid sequence from bovine cDNA. This smaller version of cytochrome b561 may contain only five transmembrane segments rather than the previously proposed six segments. The new model is in agreement with our previous results on transmembrane topology of the gene product. Northern-blot analysis shows an expanded tissue distribution of cytochrome mRNA expression where previous immunological assays were negative. These results support the hypothesis that cytochrome b561 is a marker for peptidergic and adrenergic tissues.
Cell surface-mediated activation of progelatinase A: demonstration of the involvement of the C-terminal domain of progelatinase A in cell surface binding and activation of progelatinase A by primary fibroblasts
Authors: Ward R V , Atkinson S J , Reynolds J J , Murphy G
Published: August 2015

We report that the isolated C-terminal domain of progelatinase A is inhibitory to the activation of this proenzyme by primary skin fibroblast plasma membranes but is unable to inhibit organomercurial-induced self-cleavage and activation. Ligand binding studies demonstrate that fibroblasts stimulated with concanavalin A to activate progelatinase A have a significantly enhanced level of cell surface-associated progelatinase A. Tissue inhibitor of metalloproteinases-2 (TIMP-2), an effective inhibitor of membrane-mediated progelatinase A activation, is able to abolish the enhanced level of cell surface-associated progelatinase A that occurs following stimulation. TIMP-1, a poor inhibitor of membrane activation, is unable to inhibit the cell surface binding of progelatinase A. The enhancement in the binding of 125I-progelatinase A to fibroblasts following concanavalin A stimulation can be blocked by the inclusion of excess C-terminal gelatinase A but not by a truncated form of gelatinase A lacking the C-terminal domain. Scatchard analysis of the binding of 125I-progelatinase A to concanavalin A-stimulated fibroblasts has identified 950,000 gelatinase binding sites per cell with a Kd of 1.3 x 10(-8) M. Analysis of non-stimulated fibroblasts has identified 500,000 sites per cell with a Kd of 2.6 x 10(-8) M. We propose that membrane-mediated activation of progelatinase A involves binding of the proenzyme through its C-terminal domain to the cell surface and that TIMP-2 can inhibit activation by interaction with progelatinase A through the C-terminal domain, thus preventing binding of the proenzyme.
Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein
Authors: Monaco M E , Feldman M , Kleinberg D L
Published: August 2015

Substantial purification of rat liver phosphatidylinositol (PtdIns) synthase has been achieved by a combination of Hecameg extraction, heat treatment, affinity chromatography and chromatography on PBE-94. The activity chromatographs as a single peak which has an apparent molecular mass between 150 and 200 kDa on Sepharose 4B. When analysed by SDS/PAGE, two major bands are seen. The enzyme activity is correlated with a protein band of 21 kDa. A second band, at 51 kDa, is eluted from a PBE-94 column slightly ahead of the activity. Manganese is an absolute requirement for stabilization of activity in the presence of detergent. The effect of manganese is optimal at 0.5 mM; magnesium at a concentration of 10 mM is only minimally effective. Substrate Kms are 1.3 mM and 9.5 microM for inositol and CDP-diacylglycerol respectively. The activity eluting from the PBE-94 column is purified 5000-fold over the post-mitochondrial supernatant.
Identical regional mechanisms of intracellular free Ca2+ concentration increase during polarized agonist-evoked Ca2+ response in pancreatic acinar cells
Authors: Toescu E C , Gallacher D V , Petersen O H
Published: August 2015

The initial increase of intracellular free Ca2+ concentration ([Ca2+]i) following agonist stimulation is spatially restricted to one pole of the cell, from where a wave of [Ca2+]i spreads across the cytosol. In the present study we have investigated the dynamic properties of the agonist-activated Ca(2+)-release mechanisms in different regions of the acinar cell and show that, during maximal agonist stimulation, the rate of [Ca2+]i increase at the secretory pole is identical with that recorded at the basal pole. Furthermore, the relationship between [Ca2+]i and the apparent rate of [Ca2+]i increase is similar in both regions of the cell. The data show that whereas the sensitivity to the Ca(2+)-releasing agent is different in different regions of the cell, the process of [Ca2+]i increase, once triggered, will proceed in an identical fashion, irrespective of the area of the cell.
Human blood cells support the reduction of low-density-lipoprotein-associated cholesteryl ester hydroperoxides by albumin-bound ebselen
Authors: Christison J , Sies H , Stocker R
Published: August 2015

Ebselen, a glutathione peroxidase mimic capable of reducing simple as well as complex hydroperoxides, including those of phospholipids and cholesteryl esters in intact oxidized low-density lipoprotein (LDLox), requires the presence of low-molecular-mass thiols to be active. In plasma, the drug is thought to be transported as an inactive albumin complex. As formation of LDLox is likely to occur extracellularly, we tested under which conditions ebselen can support reduction of LDLox-associated cholesteryl ester hydroperoxides outside cells. We observed that addition of albumin-bound ebselen to whole blood, but not plasma, resulted in reduction of LDLox-associated cholesteryl linoleate hydroperoxides to the corresponding hydroxides. The observed reduction was rapid and its extent increased with increasing concentrations of ebselen. Physical contact of blood cells with LDLox was not required for this reducing activity. These results demonstrate that, in the presence of blood cells, extracellular ebselen is catalytically active. They suggest that ebselen may be considered as a drug for extracellular targets.
Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions
Authors: Lobban M , Shakur Y , Beattie J , Houslay M D
Published: August 2015

In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set (‘DPD/PDE-4-common’), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid-brain (approx. 3%. The isolated cDNAs for DPD and PDE-4 appear to reflect transcription products which are expressed in vivo in brain. The unique N-terminal domain of PDE-4 is suggested to target this PDE to membranes in brain. Type-IVB PDEs are differentially expressed in various brain regions, indicating that there are tissue-specific controls on both the expression of the gene and the splicing of its products.
Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes
Authors: Baqué S , Guinovart J J , Gómez-Foix A M
Published: August 2015

The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin.
Binding of human collectins (SP-A and MBP) to influenza virus
Authors: Malhotra R , Haurum J S , Thiel S , Sim R B
Published: August 2015

Collectins are a group of soluble proteins each of which has collagenous domains and non-collagenous globular domains, the latter containing the consensus residues found in C-type lectins. Members of the collectin family are the serum proteins mannan-binding protein (MBP), conglutinin, CL-43, and the lung-associated proteins surfactant protein A (SP-A) and surfactant protein D (SP-D). MBP and conglutinin have been shown previously to bind to influenza viruses and to inhibit the infectivity and haemagglutinating activity of influenza viruses. We report here that the lung protein SP-A, like MBP, can bind to influenza virus (strain A/X31) through its lectin domain and inhibit the virus-mediated agglutination of red cells. The binding of SP-A or MBP to influenza virus was saturable, concentration-dependent, and required the presence of Ca2+ ions. Ligand-blot analysis, using MBP as ligand, of the virus lysate indicated that MBP binds to a 68 kDa viral species. The 68 kDa species was isolated to homogeneity and was shown to be the viral neuraminidase. The purified 68 kDa species inhibited the binding of both MBP and SP-A to influenza virus.
Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain
Authors: Walaas S I , Czernik A J , Olstad O K , Sletten K , Walaas O
Published: August 2015

Phospholemman, a transmembrane, 72 residue protein enriched in striated muscle and heart [Palmer, Scott and Jones (1991) J. Biol. Chem. 266, 11126-11130], is phosphorylated in response to insulin [Walaas, Horn and Walaas (1991) Biochim. Biophys. Acta 1094, 92-102]. The present study is aimed at identifying the phosphorylation sites of this protein. A synthetic peptide, GTFRSS63IRRLS68TRRR (in the single letter code) and consisting of phospholemman residues 58-72, is a substrate for both protein kinase C and cyclic AMP (cAMP)-dependent protein kinase, with Km values of 6-7 microM for both enzymes. Amino acid sequencing of the phosphopeptide shows that protein kinase C phosphorylates both Ser-63 and Ser-68, while cAMP-dependent protein kinase phosphorylates Ser-68. Thermolytic phosphopeptide mapping of 32P-labelled phospholemman from rat diaphragms shows that treatment with insulin results in labelling of phosphopeptides containing both Ser-63 and Ser-68, whereas treatment with adrenaline results in labelling of the phosphopeptide containing Ser-68. Hence, insulin and adrenaline regulate the phosphorylation of phospholemman, presumably through protein kinase C and cAMP-dependent protein kinase, respectively, on partly overlapping phosphorylation sites.
The kinetics, substrate and inhibitor specificity of the lactate transporter of Ehrlich-Lettre tumour cells studied with the intracellular pH indicator BCECF
Authors: Carpenter L , Halestrap A P
Published: August 2015

1. Suspensions of cultured Ehrlich-Lettre tumour cells were loaded with the pH-sensitive fluorescent indicator 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and changes in intracellular pH upon addition of L-lactate and other monocarboxylates were continuously monitored by fluorimetry using dual-wavelength excitation (450/500 nm) and single-wavelength emission (> 520 nm). 2. The rapid fluorescence changes were analysed by first-order regression analysis, and with suitable calibration procedures this enabled calculation of initial rates of proton uptake associated with monocarboxylate transport. 3. The stoichiometry was shown to be one proton per lactate molecule transported. 4. The kinetics of carrier-mediated transport of a wide range of monocarboxylates were determined at 25 degrees C. The Km values for L-lactate, pyruvate and D-lactate were found to be 4.54, 0.72 and 27.5 mM respectively, similar to values found previously for rat erythrocytes. This similarity was shared with a wide range of variously substituted C2, C3 and C4 monocarboxylates, all of which were transported with similar Vmax. No stereoselectivity was found in the Km values for D- and L-2-chloropropionate (0.75 mM) or D- and L-3-hydroxybutyrate (11 mM), but in the latter case the Vmax. of the D-isomer was twice that of the L-isomer. 5. The temperature-dependence of L-lactate transport demonstrated a transition point, with activation energies of 60 and 109 kJ.mol-1 above and below 19 degrees C respectively The Km for L-lactate below the transition temperature was about half that above it. 6. Inhibition of lactate transport into tumour cells by a wide range of compounds known to inhibit the erythrocyte monocarboxylate carrier was analysed. Patterns of inhibition were similar to those seen in the erythrocyte, but the Ki values were 2-4-fold higher in the tumour cells. 7. It is concluded that tumour cells contain an isoform of the monocarboxylate carrier with functional properties almost identical with that found in erythrocytes. This is probably identical with MCT1, which was recently cloned and sequenced from Chinese Hamster Ovary cells [Kim Garcia, Goldstein, Pathak, Anderson and Brown (1994) Cell 76, 865-873].
Modification of glutathione S-transferase 3-3 mutants with 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone. Identification of the C-terminal tryptic fragment as part of the H-site and evidence that 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone is not specific for cysteine labelling
Authors: Hong J L , Liu L F , Wang L Y , Tsai S P , Hsieh C H , Hsiao C D , Tam M F
Published: August 2015

A triple mutant of rat liver glutathione S-transferase 3-3 that has all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were reacted with 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GS-1,4-TCBQ). The modified proteins were analysed on a triple-quadrupole mass spectrometer equipped with an electrospray ionization source. At an enzyme: GS-1,4-TCBQ ratio of 1:10, the enzymes were modified at multiple sites. Covalent attachment of a single inhibitor on to the protein was achieved by lowering the enzyme: GS-1,4-TCBQ ratio to 1:1. Results from m.s. analyses suggest that the inhibitor on the CallSY115F mutant exists as a glutathionyl dichlorobenzoquinone derivative. The modifiers of the CallS mutants are glutathionyl monochlorobenzoquinone derivatives. Therefore, GS-1,4-TCBQ reacts at a single site on CallSY115F, but probably cross-links two regions on wild-type and CallS mutant. To confirm our observation, CallS was modified with 1-chloro2,4-dinitrobenzene, which specifically labels Tyr-115, before reacting with GS-1,4-TCBQ. The inhibitor formed a glutathionyl dichlorobenzoquinone adduct on the dinitrophenyl-CallS mutant. In addition, the benzoquinone derivative on the protein can be partially removed by 1-chloro-2,4-dinitrobenzene. Peptide mapping and sequencing analysis of the GS-1,4-TCBQ-modified CallS mutant revealed that the C-terminal 16-amino-acid fragment is labelled. Molecular modelling suggests the C(5) and C(6) on the benzoquinone ring of the inhibitor interact with the oxygen atoms of Tyr-115 and Ser-209 respectively.
Characterization of a thioredoxin-related surface protein
Authors: Dean M F , Martin H , Sansom P A
Published: August 2015

A surface-associated sulphydryl (thiol) protein (SASP) constitutively present in most nucleated cells was purified from human THP-1 monocytes and rat C6 glioma cells. The human protein was similar in mass and isoelectric point and had the same N-terminal amino acid sequence to adult T-cell leukemia-derived factor (ADF), a growth factor secreted by human lymphoid cells which is able to induce increased expression of interleukin-2 receptors. A further internal amino acid sequence, determined following cleavage of human SASP with cyanogen bromide, was also identical to the corresponding sequence deduced for ADF. Samples of SASP were able to reductively depolymerize human immunoglobulin, a property shared with thioredoxin, a ubiquitous protein, almost identical to ADF, with an essential function in many thiol-dependent reducing reactions. Furthermore, SASP purified from rat C6 glioma cells had an identical N-terminal amino acid sequence to that deduced for rat liver thioredoxin, showing that they were both members of the same family of proteins. The use of membrane-impermeable thiol reagents indicated that SASP was predominantly a cell-surface protein, and was not normally secreted. This SASP protein appeared to be a surface-associated form of thioredoxin that was constitutively present in a wide range of cells and was related to ADF, a secreted form of the same protein.
The α-5 segment of Bacillus thuringiensis δ-endotoxin: in vitro activity, ion channel formation and molecular modelling
Authors: Gazit E , Bach D , Kerr I D , Sansom M S P , Chejanovsky N , Shai Y
Published: August 2015

A peptide with a sequence corresponding to the highly conserved alpha-5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3429-3436]), was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of alpha-5 at either the N-terminal amino acid or the epsilon-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled alpha-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled alpha-5 peptides were protected from enzymic proteolysis. Functional characterization of alpha-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of alpha-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by alpha-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for alpha-5 in the toxic mechanism of delta-endotoxins, and assign alpha-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alpha-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS Microbiol. Lett. 111, 255-262].