bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation
Authors: Sahu A , Kozel T R , Pangburn M K
Published: August 2015

The specificity of the thioester-containing site in three plasma proteins is regulated by elements of their protein structures other than the thioester bond itself. Human C4A and alpha 2-macroglobulin preferentially form amide linkages while human C3 primarily forms ester linkages with hydroxyl groups. We have examined the thioester in C3 and found evidence of strong preferences for certain carbohydrates, indications of selectivity for specific positions on those carbohydrates and a preference for terminal sugars in polysaccharides. A testable set of rules are derived from these findings which predict preferred attachment sites on polysaccharides. A computer model of the effect of different reactivities on activation of the alternative pathway of complement suggested that organisms might greatly alter their susceptibility to complement with small changes in carbohydrate structure. While a random selection of 20 biological particles showed no correlation between activation and C3b attachment efficiency, subsets of related organisms differing primarily in their surface polysaccharide exhibited stronger correlations. The strongest correlation occurred in a series of the yeasts (Cryptococcus neoformans) possessing capsular polysaccharides with one, two, three or four branching xylose sugars per repeating unit. These organisms exhibited capture efficiencies for metastable C3b from 12% (one-xylose strain) to 41% (four-xylose strain).
Structural and functional characterization of the rod outer segment membrane guanylate cyclase
Authors: Goraczniak R M , Duda T , Sitaramayya A , Sharma R K
Published: August 2015

In the vertebrate photoreceptor cell, rod outer segment (ROS) is the site of visual signal-transduction process, and a pivotal molecule that regulates this process is cyclic GMP. Cyclic GMP controls the cationic conductance into the ROS, and light causes a decrease in the conductance by activating hydrolysis of the cyclic nucleotide. The identity of the granylate cyclase (ROS-GC) that synthesizes this pool of cyclic GMP is unknown. We now report the cloning, expression and functional characterization of a DNA from bovine retina that encodes ROS-GC.
Functional characterization of two human sulphotransferase cDNAs that encode monoamine- and phenol-sulphating forms of phenol sulphotransferase: substrate kinetics, thermal-stability and inhibitor-sensitivity studies
Authors: Veronese M E , Burgess W , Zhu X , McManus M E
Published: August 2015

The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated by us from liver and brain, respectively [Zhu, Veronese, Sansom, and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.
Regulation of splicing is responsible for the expression of the muscle-specific 2a isoform of the sarco/endoplasmic-reticulum Ca2+-ATPase
Authors: Van den Bosch L , Eggermont J , De Smedt H , Mertens L , Wuytack F , Casteels R
Published: August 2015

Tissue-specific alternative processing of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) transcripts generates functionally different Ca2+ pump isoforms in muscle compared with non-muscle tissues. In non-muscle cells, the SERCA2 pre-mRNA can be polyadenylated at a site located between the donor and acceptor splice site of an intron which is only removed in muscle tissues. To define the cis-active elements involved in differential processing, we constructed a minigene (pCM beta SERCA2) containing the 3′ end of the SERCA2 gene. When stably transfected into a myogenic cell line, minigene transcripts were differentially processed depending on the differentiation state of the cells. This proves that the essential elements required for regulated processing are present in the construct. Furthermore, co-transfection of the pCM beta SERCA2 minigene and a myogenin expression vector in a fibroblast cell line induced muscle-specific splicing of transcripts from pCM beta SERCA2. This shows that trans-acting factor(s) responsible for muscle-specific processing can be induced by one of the important regulatory genes of muscle differentiation. Inactivation of the non-muscle poly(A) site did not induce splicing in non-muscle cells. This excludes a simple competition model between splicing and polyadenylation, but it is consistent with splicing being very inefficient in non-muscle cells. Moreover, splicing could be induced in non-muscle cells by optimizing the muscle-specific donor splice site and/or by shortening the intron length. We therefore propose that expression of the muscle-specific SERCA2a isoform is the result of activation of an otherwise inefficient splicing process.
CCAAT/enhancer-binding proteins α and β in brown adipose tissue: evidence for a tissue-specific pattern of expression during development
Authors: Manchado C , Yubero P , Viñas O , Iglesias R , Villarroya F , Mampel T , Giralt M
Published: August 2015

CCAAT/enhancer-binding protein (C/EBP) alpha mRNA and its protein products C/EBP alpha and 30 kDa C/EBP alpha are expressed in rat brown-adipose tissue. Results also demonstrate the expression of C/EBP beta mRNA and its protein products C/EBP beta and liver inhibitory protein (LIP) in the tissue. The abundance of C/EBP alpha and C/EBP beta proteins in adult brown fat is similar to that found in adult liver. However, the expression of C/EBP alpha and C/EBP beta is specifically regulated in brown fat during development. C/EBP alpha, 30 kDa C/EBP alpha, C/EBP beta and LIP content is several-fold higher in fetal brown fat than in the adult tissue, or liver at any stage of development. Peak values are attained in late fetal life, in concurrence with the onset of transcription of the uncoupling protein (UCP) gene, the molecular marker of terminal brown-adipocyte differentiation. When adult rats are exposed to a cold environment, which is a physiological stimulus of brown-adipose tissue hyperplasia and UCP gene expression, a specific rise in C/EBP beta expression with respect to C/EBP alpha, 30 kDa C/EBP alpha and LIP is observed. Present data suggest that the C/EBP family of transcription factors has an important role in the development and terminal differentiation of brown-adipose tissue.
Human inositol 1,4,5-trisphosphate type-1 receptor, InsP3R1: structure, function, regulation of expression and chromosomal localization
Authors: Yamada N , Makino Y , Clark R A , Pearson D W , Mattei M G , Guénet J L , Ohama E , Fujino I , Miyawaki A , Furuichi T , MIKOSHIBA K
Published: August 2015

We have isolated cDNA clones encoding an inositol 1,4,5-trisphosphate receptor type 1 (InsP3R1) from human uteri and a leukaemic cell line, HL-60. Northern-blot analysis showed that approx. 10 kb of InsP3R1 mRNA is expressed in human uteri, oviducts and HL-60 cells. The predicted amino acid sequence of human InsP3R1 (2695 amino acids) has 99% identity with that of the mouse SI-/SII- splicing counterpart. Western-blot analysis with anti-(mouse InsP3R1) antibodies showed that InsP3R1 protein of human uteri and oviducts of approx 220 kDa is immunostained. Northern-blot analysis of HL-60 cell differentiation along the neutrophilic lineage induced by retinoic acid or dimethylsulphoxide showed an accompanying enhanced expression of InsP3R1 mRNA. Immunohistochemical analysis of the cerebella of spinocerebellar degeneration patients showed a variable loss of Purkinje cells with an altered pattern of immunostaining. The InsP3R1 gene (Insp3r1) was localized to the 3P25-26 region of human chromosome 3. The data presented here clearly show that InsP3R1 exists widely in human tissues and may play critical roles in various kinds of cellular functions.
Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase
Authors: Chapman-Smith A , Turner D L , Cronan J E , Morris T W , Wallace J C
Published: August 2015

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.
Interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide T669
Authors: Kracht M , Shiroo M , Marshall C J , Hsuan J J , Saklatvala J
Published: August 2015

We have isolated from KB cells stimulated with interleukin-1 (IL-1) a protein kinase that phosphorylates a peptide (T669) based on the sequence around T669 of the epidermal growth factor (EGF) receptor. The enzyme, which had an apparent molecular mass of 45 kDa on gel-filtration chromatography, was purified 170,000-fold from cytosolic extracts by sequential chromatography on Mono Q, Mono S, phenyl-Sepharose, Superose 12, ATP-Sepharose and Mono Q. The enzyme activity co-chromatographed at the last step with a 45 kDa protein band that stained for phosphotyrosine. This peak fraction also contained some actin and a 60 kDa protein that stained weakly for phosphotyrosine. The T669 peptide is a substrate for mitogen-activated protein (MAP) kinase. Amounts of IL-1-induced T669 kinase and activated recombinant p42 MAP kinase having equal activity on T669 peptide were compared on commonly used MAP kinase substrates. T669 kinase was two or three orders of magnitude less active on myelin basic protein or microtubule-associated protein-2 than was MAP kinase. The IL-1-induced T669 kinase did not react with antiserum to p42/p44 MAP kinase. It was inactivated by treatment with protein phosphatase 2A or protein phosphotyrosine phosphatase 1B, so it may be regulated by dual phosphorylation in similar fashion to MAP kinase. The dephosphorylated enzyme was not re-activated by MAP kinase kinase. This novel enzyme could lie on a kinase cascade induced by IL-1. It may be responsible for phosphorylating T669 of the EGF receptor.
Elevated intracellular cyclic AMP exerts different modulatory effects on cytosolic free Ca2+ oscillations induced by ADP and ATP in single rat hepatocytes
Authors: Green A K , Cobbold P H , Dixon C J
Published: August 2015

Single aequorin-injected hepatocytes respond to agonists acting via the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]free). The duration of [Ca2+]free transients is characteristic of the stimulating agonist. We have previously reported that ADP and ATP, which are believed to act through a single P(2y)-purinoceptor species, induce very different oscillatory [Ca2+]free responses in the majority of hepatocytes. We have interpreted these data as evidence for two separate Ca(2+)-mobilizing purinoceptors for these nucleotides. We show here that the elevation of intracellular cyclic AMP concentration, by the co-application of either dibutyryl cyclic AMP or 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)butyryl]- forskolin (L858051), exerts different modulatory effects on [Ca2+]free oscillations induced by ADP and ATP in single rat hepatocytes. Elevated intracellular cyclic AMP levels enhance the frequency and peak [Ca2+]free of transients induced by ADP. In contrast, the elevation of intracellular cyclic AMP levels in hepatocytes producing [Ca2+]free oscillations in response to ATP stimulates either an increase in the duration of transients or a sustained rise in [Ca2+]free. The data illustrate a further difference between the oscillatory [Ca2+]free responses of hepatocytes to ADP and ATP, thus further arguing against ADP and ATP acting via a single purinoceptor species.
Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum
Authors: Ganguly T C , Liu Y , Hyde J F , Hagenbuch B , Meier P J , Vore M
Published: August 2015

We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity.
Inhibition of electron flow through complex I of the mitochondrial respiratory chain of Ehrlich ascites carcinoma cells by methylglyoxal
Authors: Ray S , Dutta S , Halder J , Ray M
Published: August 2015

The effect of methylglyoxal on the oxygen consumption of Ehrlich-ascites-carcinoma (EAC)-cell mitochondria was tested by using different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal on the oxygen consumption by these cells. The results indicate that methylglyoxal strongly inhibits ADP-stimulated alpha-oxo-glutarate and malate plus pyruvate-dependent respiration, whereas, at a much higher concentration, methylglyoxal fails to inhibit succinate-dependent respiration. Methylglyoxal also fails to inhibit respiration which is initiated by duroquinol, an artificial electron donor. Moreover, methylglyoxal cannot inhibit oxygen consumption when the NNN'N′-tetramethyl-p-phenylenediamine by-pass is used. The inhibitory effect of methylglyoxal is identical on both ADP-stimulated and uncoupler-stimulated respiration. Lactaldehyde, a catabolite of methylglyoxal, can exert a protective effect on the inhibition of EAC-cell mitochondrial respiration by methylglyoxal. We suggest that methylglyoxal possibly inhibits the electron flow through complex I of the EAC-cell mitochondrial respiratory chain.
Major proteins of bovine seminal plasma inhibit phospholipase A2
Authors: Manjunath P , Soubeyrand S , Chandonnet L , Roberts K D
Published: August 2015

We have recently shown that the major proteins of bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called BSP proteins) bind to spermatozoa and that the binding sites on the plasma membrane of spermatozoa are choline phospholipids. In view of the fact that these phospholipids are substrates for phospholipase A2 (PLA2), a key enzyme in sperm capacitation and the acrosome reaction, the effect of BSP proteins on this enzyme activity was investigated. Since these BSP proteins are ubiquitous, the effect on pig pancreatic PLA2 was also studied. In contrast with control proteins, when preincubated with phosphatidylcholine as substrate, all BSP proteins inhibited both pancreatic and sperm PLA2 activity in a dose-dependent manner and in the presence of 1-6 microM BSP protein the enzyme activity was completely abolished. When phosphatidylethanolamine was used as substrate, only pancreatic PLA2 was inhibited. On the other hand, when the BSP proteins were preincubated with the enzyme followed by addition of substrate, a biphasic effect was observed; there was stimulation of enzyme activity below 1.3 microM BSP followed by an inhibition above this concentration. The inhibitory activity was trypsin-sensitive but heat-resistant. The effect of co-incubation of heparin, which is implicated in sperm capacitation and which also interacts with BSP proteins, was studied. Heparin (10 microM) had no effect on the PLA2 inhibitory activity exhibited by all BSP proteins. The PLA2 inhibitory effect exhibited by BSP proteins was abolished with excess substrate. The BSP proteins were adsorbed on PLA2-agarose and could be affinity cross-linked to the enzyme, indicating a direct interaction of enzyme with the inhibitor. These results suggest that these BSP proteins modulate PLA2 activity and therefore, phospholipid metabolism.
Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells
Authors: Garland A M , Grady E F , Payan D G , Vigna S R , Bunnett N W
Published: August 2015

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.
Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line
Authors: Chang T C , Wang J K , Hung M W , Chiao C H , Tsai L C , Chang G G
Published: August 2015

Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA transcript in choriocarcinoma cells. Analysis of the degradation of BC-M1 ALP mRNA showed a similar half-life of 27 h in the untreated and in dexamethasone- or RA-treated cells. These findings demonstrated that the induction of ALP in BC-M1 cells by dexamethasone is mainly due to the increase in the transcription of the ALP gene.
Partial purification and reconstitution of the sarcolemmal l-lactate carrier from rat skeletal muscle
Authors: Allen P J , Brooks G A
Published: August 2015

Purified sarcolemmal membranes from mixed rat hindlimb muscle were solubilized with octylglucoside and the extract subjected to hydroxylapatite (HA) chromatography. Following protein elution with a sodium phosphate gradient and detergent removal by dialysis, the HA eluate was reconstituted into asolectin liposomes using a freeze-thaw procedure. Specific L-[14C]lactate transport activity eluting from the 0.2 M sodium phosphate fraction was 30-fold higher compared with native sarcolemmal vesicles (31.64 versus 1.06 nmol/min per mg). The reconstituted carrier exhibited Michaelis-Menten saturation kinetics with Km and Vmax. values of 46.2 +/- 6.6 mM and 498.7 +/- 17.2 nmol/15 s per mg respectively. L-Lactate transport activity was inhibited 57% by preincubation of proteoliposomes with 10 mM alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. Analysis of the HA eluates by SDS/PAGE showed the presence of a 34 kDa band corresponding to lactate transport activity. Reconstitution of lactate transport activity eluting from the HA column, together with SDS/PAGE analysis suggests the presence of a 34 kDa polypeptide mediating sarcolemmal lactate exchange in rat skeletal muscle.
Zinc, vanadate and selenate inhibit the tri-iodothyronine-induced expression of fatty acid synthase and malic enzyme in chick-embryo hepatocytes in culture
Authors: Zhu Y , Goodridge A G , Stapleton S R
Published: August 2015

Insulin regulates the expression of genes involved in a variety of metabolic processes. In chick-embryo hepatocytes in culture, insulin amplifies the tri-iodothyronine (T3)-induced enzyme activity, and the level and rate of transcription of mRNA for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone, however, has little or no effect on the expression of these genes. In chick-embryo hepatocytes, the mechanism by which insulin regulates the expression of these or other genes is not known. Several recent studies have compared the effects of zinc, vanadate and selenate on insulin-sensitive processes in an attempt to probe the mechanism of insulin action. Because zinc, vanadate and selenate mimic the effects of insulin on several processes, they have been termed insulin-mimetics. We have studied the effect of zinc, vanadate and selenate on the T3-induced expression of both FAS and ME. Like insulin, these agents had little or no effect on the basal activities for FAS and ME in chick-embryo hepatocytes in culture for 48 h. Unlike insulin, however, zinc, vanadate and selenate inhibited the T3-induced activities and mRNA levels of both FAS and ME. Maximal inhibition was achieved at concentrations of 50 microM zinc or vanadate, or 20 microM selenate. Zinc and vanadate also inhibited the T3-induced transcription of the FAS and ME genes. Although the mechanism of this inhibition is unknown, our results indicate that it is not mediated through inhibition of binding of T3 to its nuclear receptor nor through a general toxic effect. Thus zinc, vanadate and selenate are not insulin-mimetics under all conditions, and their effects on other insulin-sensitive processes may be fortuitous and unrelated to actions or components of the insulin signalling pathway.
Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum
Authors: Rogers M J , Ji X , Russell R G G , Blackburn G M , Williamson M P , Bayless A V , Ebetino F H , Watts D J
Published: August 2015

Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.
Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor- and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts
Authors: Andrieu N , Salvayre R , Levade T
Published: August 2015

The hydrolysis of sphingomyelin (SPM) has been reported to mediate a number of responses to extracellular agents, including cytokines. The so-called SPM cycle may result from the activation of different types of sphingomyelinases (SPMases). We investigated the hypothetical contribution of acid lysosomal SPMase in the SPM signal-transduction pathway. We examined the ability of human skin fibroblasts with a genetic deficiency of acid lysosomal SPMase activity to respond to tumour necrosis factor alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). We report that both cytokines promoted SPM hydrolysis in fibroblasts derived from patients with Niemann-Pick disease or I-cell disease, similar to that observed in normal cells. Treatment of normal fibroblasts with cationic amphiphilic drugs resulted in inhibition of acid SPMase activity, but had no effect on cytokine-induced SPM turnover. In addition, TNF-alpha and IL-1 beta stimulated [3H]thymidine incorporation in Niemann-Pick fibroblasts, as in normal cells. Thus our results argue against a role for acid endolysosomal SPMase in mediating the cytokine-induced SPM signalling cascade.
The role of hypusine depletion in cytostasis induced by S-adenosyl-l-methionine decarboxylase inhibition: new evidence provided by 1-methylspermidine and 1,12-dimethylspermine
Authors: Byers T L , Lakanen J R , Coward J K , Pegg A E
Published: August 2015

The abilities of the natural polyamines, spermidine and spermine, and of the synthetic analogues, 1-methylspermidine and 1,12-dimethylspermine, to reverse the effects of the S-adenosyl-L-methionine decarboxylase inhibitor 5′-([(Z)-4-aminobut-2-enyl]methylamino)-5′-deoxyadenosine (AbeAdo) on L1210-cell growth were studied. L1210 cells were exposed to AbeAdo for 12 days to induce cytostasis and then exposed to spermidine, spermine, 1-methylspermidine or 1,12-dimethylspermine in the continued presence of AbeAdo. AbeAdo-induced cytostasis was overcome by the natural polyamines, spermidine and spermine. The cytostasis was also reversed by 1-methylspermidine. 1,12-Dimethylspermine had no effect on the AbeAdo-induced cytostasis of chronically treated cells, although it was active in permitting growth of cells treated with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The initial 12-day exposure to AbeAdo elevated intracellular putrescine levels, depleted intracellular spermidine and spermine, and resulted in the accumulation of unmodified eukaryotic translation initiation factor 5A (eIF-5A). Exposure of these cells to exogenous spermidine, which is the natural substrate for deoxyhypusine synthase, resulted in a decrease in the unmodified eIF-5A content. 1-Methylspermidine, which was found to be a substrate of deoxyhypusine synthase in vitro, also decreased the levels of unmodified eIF-5A in the AbeAdo-treated cells. Although spermine is not a substrate of deoxyhypusine synthase, spermine was converted into spermidine in the L1210 cells, and spermine addition to AbeAdo-treated cells resulted in the appearance of both intracellular spermine and spermidine and in the decrease in unmodified eIF-5A. Exogenous 1,12-dimethylspermine, which was not metabolized to spermine or to 1-methylspermidine and was not a substrate of deoxyhypusine synthase in vitro, did not decrease levels of unmodified eIF-5A. The finding that AbeAdo-induced cytostasis was only reversed by polyamines and polyamine analogues that result in the formation of hypusine or an analogue in eIF-5A is consistent with the hypothesis [Byers, Wiest, Wechter and Pegg (1993) Biochem. J. 290, 115-121] that AbeAdo-induced cytostasis is due to the depletion of the hypusine-containing form of eIF-5A, which is secondary to the depletion of spermidine by inhibition of S-adenosyl-L-methionine decarboxylase.
Expression of mouse phosphofructokinase-M gene alternative transcripts: evidence for the conserved two-promoter system
Authors: Nakajima H , Noguchi T , Hamaguchi T , Tomita K , Hanafusa T , Kono N , Tanaka T , Kuwajima M , Matsuzawa Y
Published: August 2015

Molecular cloning of the 5′ part of mouse phosphofructokinase-M cDNA was performed. In the 46 cDNA clones isolated, there were two classes of 5′ untranslated sequences. One had an EcoRI site within its 5′ untranslated sequence. This showed 83.0% similarity with human type B mRNA for phosphofructokinase-M. The other lacked an EcoRI site, showing 92.9% similarity with human type C mRNA. Using the reverse-transcription PCR technique, we found that the transcript with an EcoRI site was exclusively expressed in cardiac and skeletal muscles, while that without an EcoRI site was expressed in all the mouse tissues examined. The results suggested that the mouse phosphofructokinase-M gene was transcribed through alternative splicing by the multiple promoters. This transcription mechanism was considered to be evolutionarily conserved. The level of phosphofructokinase-M gene expression in mouse cardiac and skeletal muscles decreased in the ketotic diabetic state. Although the regulatory mechanism and the physiological significance are not fully known, this would indicate that phosphofructokinase-M gene transcripts are affected during the diabetic state.
Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade
Authors: Hill C S T , Oh S Y , Schmidt S A , Clark K J , Murray A W
Published: August 2015

Lysophosphatidic acid (LPA) was shown to be a powerful inhibitor of gap-junctional communication between cultured rat liver WB cells, as determined by the transfer of Lucifer Yellow, with 50% inhibition obtained at about 0.3 microM LPA. Inhibition of communication was rapid (5 min) and was maintained for at least 80 min. After incubation for 3 h with LPA, communication competence was partially restored and dye transfer was refractory to further addition of LPA. Communication in LPA-refractory cells retained sensitivity to inhibition by phorbol ester and by epidermal growth factor (EGF). LPA-induced inhibition was associated with phosphorylation of connexin-43 protein, as detected by slower migration of the protein detected on Western blots, which could be eliminated by incubation of samples with alkaline phosphatase. A close correspondence was observed between the time- and dose-dependency of LPA effects on communication and the induction of mitogen-activated protein kinase (MAP kinase). Activation of both the 42 kDa and 44 kDa subspecies were confirmed by mobility shifts on Western blots using an anti-(MAP kinase R1) (erk 1-III) antibody and by fractionation on Mono Q columns. Cells pretreated with phorbol ester for 24 h were insensitive to phorbol ester inhibition of communication or activation of MAP kinase, but retained their sensitivity to LPA. The results indicate that LPA initiates the activation of protein kinase cascades in WB cells that are probably independent of protein kinase C and identifies connexin-43 as one substrate for the activated kinases.
A study on the functional subunits of phospholipases A2 by enzyme immobilization
Authors: Ferreira J P M , Sasisekharan R , Louie O , Langer R
Published: August 2015

Pancreatic and venom phospholipases A2 have complex and distinct oligomerization behaviour. Pancreatic enzymes are monomeric in solution, but their quaternary structure at interfaces is unknown. On the other hand, certain crotalid venom phospholipases A2 are dimeric in solution, and different reports have proposed either the monomer or the dimer as the catalytically functional subunit. In this study, enzyme immobilization was used as a tool for determining the functional subunits of these enzymes. The dimeric Crotalus atrox phospholipase A2 was covalently attached to agarose beads, via either the amine or the carboxylic groups of the protein. In the first case immobilization led to an 80% loss of activity as compared with the soluble form, and measured by using micellar diheptanoylphosphocholine. Inclusion of micellar protectants in the coupling media did not improve the activity. Enzyme immobilized via carboxylic groups was 2-3-fold more active than the amine-coupled form. In a second approach, Crotalus atrox enzyme was immobilized with single-subunit attachment. The removal, with denaturating washes, of the non-covalently bound units involved in monomer-monomer interactions, caused a large decrease in specific activity of the support-bound enzyme. This suggests the dimeric form as the fully active one. Similar procedures were also carried out with pig pancreatic and Naja naja phospholipases A2. The results indicated that these enzymes are active as monomers.
Chemotactic peptide down-regulation of calcium mobilization induced by platelet-activating factor and by leukotriene B4 in human neutrophils is uncovered by protein phosphatase inhibitors
Authors: Montero M , Garcia-Sancho J , Alverez J
Published: August 2015

When human neutrophils were incubated in the presence of the protein phosphatase inhibitors calyculin A or okadaic acid, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) produced a sustained (> 5 min) inhibition of the Ca2+ mobilization from intracellular stores induced by platelet-activating factor (PAF) or by leukotriene B4 (LTB4). No effect on Ca2+ mobilization by PAF or LTB4 was observed 2 min after the addition of fMLP alone or only in the presence of phosphatase inhibitors, but a similar inhibition was produced by high (> 50 nM) concentrations of phorbol 12,13-dibutyrate (PDB). However, inhibition by PDB was sensitive to the protein kinase C (PKC) inhibitors staurosporin and Ro 31-8220, while inhibition by fMLP and calyculin A was not. These results suggest that fMLP induces a transient phosphorylation not mediated by PKC which interferes at some point with the transduction pathway leading from the plasma membrane receptors for PAF and LTB4 to the release of Ca2+ from the stores. Protein phosphatases 1 and/or 2A revert the inhibition effected by fMLP within less than 2 min. PAF and LTB4 were also able to activate this mechanism to a smaller extent. Phosphatase inhibitors also delayed by 1-2 s the start of agonist-induced rises in [Ca2+]i, and this delay was further increased by previous addition of any other agonist. Finally, given that both phosphatase inhibitors and low concentrations of PDB (2-10 nM) strongly inhibit Ca2+ entry, we conclude that phosphorylation down-regulates both agonist-induced Ca2+ entry and Ca2+ mobilization, but with different potency.
Archaebacterial histone-like protein MC1 can exhibit a sequence-specific binding to DNA
Authors: Teyssier C , Laine B , Gervais A , Maurizot J C , Culard F
Published: August 2015

The binding of MC1 protein, the major chromosomal protein of the archaebacterium Methanosarcina sp. CHTI 55, to the region preceding the strongly expressed genes encoding methyl coenzyme reductase in a closely related micro-organism has been investigated. By gel retardation and DNAase I footprinting assays, we identified a preferential binding sequence in an open reading frame of unknown function. The large area of DNA protected against DNAase I is interrupted by a strong cleavage enhancement site on each strand. By circular permutation assays, we showed that the DNA bends upon MC1 binding. Furthermore we observed that the presence of a sequence outside the binding site can induce an unusual electrophoretic behaviour in some complexes.
Mechanism of interaction between actin and membrane lipids: a pressure-tuning infrared spectroscopy study
Authors: Gicquaud C , Wong P
Published: August 2015

Using pressure-tuning Fourier transform infrared spectroscopy to study an in vitro system consisting of actin and distearoyl-phosphatidylcholine (DSPC) liposomes, we have determined the mechanism of interaction between actin and membrane lipids. This interaction results in a significant conformational change in actin molecules. Analysis of the amide I band of actin shows an increase in the beta-sheets to alpha-helix ratio, in random turns, and in interactions between actin monomers. In the absence of lipids, the actin molecules are denatured by pressures of 8 x 10(8) Pa and more, which give rise to a random organization of the peptide chain. However, in the presence of DSPC liposomes, pressure greater than 2 x 10(8) Pa induces a change in actin conformation, which is dominated by strongly interacting beta-sheets. As the spectra of the lipid molecules are not changed by the presence of actin, the organization of the lipid molecules in the bilayer is not affected by the protein. It is concluded from these results that this interaction of actin with membrane lipids involves very few lipid molecules. These lipid molecules may interact with actin at a few specific sites on the protein.
Mitogenic function of human procathepsin D: the role of the propeptide
Authors: Fusek M , Vetvicka V
Published: August 2015

The proform of cathepsin D is secreted by some human breast-cancer cell lines upon stimulation with oestrogen. In these cell lines, procathepsin D was described to act as an autocrine mitogen, and a correlation between the cathepsin D concentration in tumour tissues and poor prognosis for the patient was demonstrated in several independent investigations. In the present study, we focused on the mechanism of procathepsin D mitogenic activity. Procathepsin D isolated from secretions of ZR-75-1 breast-cancer cell line was used to test for mitogenic activity on a set of seven human cell lines. For nanomolar procathepsin D concentrations, we found a stronger dose-responsive cellular reaction in the case of several different human breast-cancer-derived cell lines. The mitogenic activity was not blocked by the inhibition of proteolytic activity nor by the inhibition of the interaction of procathepsin D with mannose-6-phosphate receptors. On the other hand, the addition of antibodies raised against the propeptide impaired the mitogenic activity of procathepsin D, and a synthetic peptide alone corresponding to the propeptide of procathepsin D produced similar effects, as did the zymogen molecule. The synthetic propeptide was shown to block partially the interaction of procathepsin D with the cellular surface. Our results indicate that the mitogenic function involves the propeptide of cathepsin D, which appears to be recognized by a surface receptor.
Erythrocyte catalase inactivation (H2O2 production) by ascorbic acid and glucose in the presence of aminotriazole: role of transition metals and relevance to diabetes
Authors: Ou P , Wolff S P
Published: August 2015

Erythrocytes exposed to ascorbic acid in the presence of aminotriazole undergo a dose- and time-dependent inactivation of endogenous catalase which is proportional to environmental hydrogen peroxide (H2O2) concentrations. The production of H2O2 seems to be dependent upon the availability of transition metal chelatable by o-phenanthroline (OPT), although the kinetics of catalase inactivation and H2O2 production by externally added copper ions in the presence of OPT is complex. Furthermore, although glucose is also able to undergo a transition-metal-catalysed oxidation yielding H2O2, the production of H2O2 by glucose seems to be a minor process by comparison with ascorbic acid oxidation. Indeed, on the basis of these data, transition-metal-catalysed ascorbic acid oxidation is likely to be a more important source of oxidative stress in the diabetic state than hyperglycaemia.
Localization of transglutaminase-reactive glutamine residues in bovine osteopontin
Authors: Sørensen E S , Rasmussen L K , Møller L , Jensen P H , Højrup P , Petersen T E
Published: August 2015

Here we report the identification of two transglutaminase-reactive glutamines (Gln-34 and Gln-36) in bovine osteopontin (OPN). Sequence alignment revealed that these glutamines are conserved in all known OPN sequences, indicating a functional importance of this region of the protein. Furthermore, immunological analysis of bovine bone demonstrated that OPN is present in high-molecular-mass complexes in vivo. These findings support the functional aspects of a transglutaminase-catalysed cross-linking of OPN in facilitating cellular attachment and tissue calcification.
Novel K+-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch
Authors: Martin B M , Ramirez A N , Gurrola G B , Nobile M , Prestipino G , Possani L D
Published: August 2015

Two novel toxins were purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, using an immunoassay based on antibodies raised against noxiustoxin (NTX), a known K(+)-channel-blocker-peptide. The primary structure of C. l. limpidus toxin 1 was obtained by Edman degradation and was shown to be composed of 38 amino acid residues, containing six half-cystines. The first 36 residues of C. l. limpidus toxin 2 were also determined. Both toxins are capable of displacing the binding of radio-labelled NTX to rat brain synaptosomes with high affinity (about 100 pM). These toxins are capable of inhibiting transient K(+)-currents (resembling IA-type currents), in cultured rat cerebellar granule cells. About 50% of the peak currents are reduced by application of a 1.5 microM solution of toxins 1 and 2 The K+ current reduction is partially reversible, under washing but not voltage-dependent. Comparison of the primary structure of C. l. limpidus toxin 1 with other known toxins shows 74% identity with margatoxin, 64% with NTX, 51% with kaliotoxin, 39% with iberiotoxin, 37% with charybdotoxin and Lq2, and 29% with leirutoxin 1. The only invariant amino acids in all these toxins are the six cysteines, a glycine in position 26 and two lysines at positions 28 and 33, respectively. The relevance of these differences in terms of possible structure-function relationships is discussed.