bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunochemical characterization of l-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues
Authors: Boivin D , Bilodeau D , Béliveau R
Published: August 2015

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.
Microsomal triglyceride transfer protein activity remains unchanged in rat livers under conditions of altered very-low-density lipoprotein secretion
Authors: Brett D J , Pease R J , Scott J , Gibbons G F
Published: August 2015

Microsomal triglyceride transfer protein (MTP) is a heterodimeric protein consisting of a unique 97 kDa subunit and protein disulphide isomerase, that mediates the transfer of lipid between membranes and nascent lipoproteins. Mutations in the gene encoding the 97 kDa subunit of the protein cause the rare autosomal recessive disorder abetalipoproteinaemia. Recent findings in cultured cells indicate that the 5′ promoter region contains an insulin-responsive element, suggesting that the gene is responsive to insulin regulation. In this study we examined two cases where very-low-density lipoprotein (VLDL) secretion is markedly reduced: the streptozotocin-diabetic rat and 10-day-old suckling rats. In both cases MTP activity was unaltered compared with that in control animals. Equal levels of MTP were also apparent in the livers of all groups of animals, as measured by immunoblotting with an anti-MTP antiserum. These findings indicate that factors other than MTP activity are responsible for the reduction in hepatic VLDL triglyceride secretion observed in the suckling and diabetic animals.
Kinetic investigation of the action of hyaluronidase on hyaluronan using the Morgan-Elson and neocuproine assays
Authors: Vercruysse K P , Lauwers A R , Demeester J M
Published: August 2015

In this paper we describe kinetic investigations of the action of testicular hyaluronidase on hyaluronan. We have compared the use of two spectrophotometric assays, the first based on the Morgan-Elson reaction and the second on the neocuproine reaction. With the neocuproine reaction Km was found to be 0.46 mg/ml and Vmax to be 126 nmol l-1 s-1. Because of a low sensitivity and the production of interfering precipitates, the Morgan-Elson assay cannot be used for kinetic investigation of the enzyme. Furthermore this assay is prone to interference from compounds such as disodium cromoglycate, (+)-catechine, penicillamine, CaCl2 and acetate buffer.
Glutamate stably enhances the activity of two cytosolic forms of phospholipase A2 in brain cortical cultures
Authors: Kim D K , Rordorf G , Nemenoff R A , Koroshetz W J , Bonventre J V
Published: August 2015

The mechanisms by which glutamatergic neurotransmitters modulate neuronal lipid metabolism are not well established. We have directly measured phospholipase A2 (PLA2) enzymic activity in cell-free extracts from cortical neuronal cultures from rat brain and have found that the PLA2 activity is up-regulated after cells are exposed to glutamate. Brief exposure to a calcium ionophore or phorbol 12-myristate 13-acetate (PMA) stably enhanced PLA2 activity. Down-regulation of protein kinase C activity partially blocked glutamate's effects. Two Ca(2+)-and pH-dependent forms of PLA2 were identified in cytosolic extracts. Activation of both forms of PLA2 was enhanced by prior exposure of the cultures to glutamate. One of the two forms had chromatographic characteristics on heparin-Sepharose, Mono Q and Superose 12 columns similar to the 100 kDa cytosolic PLA2 (cPLA2), and was recognized by an antibody raised to pig spleen cPLA2. The second form was similar in size to Group-I and -II PLA2s but differed in chromatographic characteristics. It was not inhibited by dithiothreitol, and did not react with antibodies to pancreatic Group-I PLA2, features that distinguish it from Group-I and -II PLA2. In extracts from cells pretreated with glutamate, the activity-Ca2+ concentration dose-response relationship of the 13.5 kDa form of PLA2 was shifted to the left with activation at lower Ca2+ concentration as the result of stable modification of the enzyme induced by glutamate. Thus glutamate-induced stable enhancement of PLA2 activity, by processes involving calcium and protein kinase C activation, is a potential molecular switch probably mediating changes in synaptic function and contributing to excitotoxicity.
A novel protein, amyloid precursor-like protein 2, is present in human brain, cerebrospinal fluid and conditioned media
Authors: Webster M T , Groome N , Francis P T , Pearce B R , Sherriff F E , Thinakaran G , Felsenstein K M , Wasco W , Tanzi R E , Bowen D M
Published: August 2015

A monoclonal antibody, 3B11, was raised to a novel protein, amyloid precursor-like protein 2, which did not recognize amyloid precursor protein. Multiple bands were detected in human brain fractions and cell lysate by Western blotting, indicating the presence of isoforms, 3B11 immunoreactivity was also detected in cerebrospinal fluid and conditioned medium, indicating that the protein is secreted. Immunocytochemistry revealed 3B11 immunoreactivity in sections of human brain.
Phorbol 12-myristate 13-acetate-mediated signalling in murine bone marrow cells
Authors: Visnjić D , Batinić D , Lasić Z , Knotek M , Marusić M , Banfić H
Published: August 2015

Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence the 1,2-diacylglycerol or cyclic AMP concentrations, the intracellular Ca2+ concentration or phospholipase D activity. PMA increased particulate phospholipase A2 (PLA2) activity, lysophosphatidylcholine formation and arachidonic acid release from bone marrow cells; these effects were abolished when cells were pretreated with the putative PLA2 inhibitors heparin and mepacrine. While indomethacin and nordihydroguaiaretic acid inhibited either the cyclo-oxygenase or lipoxygenase pathway of arachidonic acid metabolism, as measured by their products prostaglandin E2 and leukotriene B4, they did not influence PMA-mediated PLA2 activation or translocation of protein kinase C (PKC) from the soluble to the particulate fraction. Treatment of cells with PMA increased the amounts of membrane-bound alpha, beta, delta, epsilon and zeta isoforms of PKC in bone marrow cells. Pretreatment of cells with PLA2 inhibitors reduced the amount of membrane-bound PKC-zeta in unstimulated cells and diminished PMA-induced translocation of PKC-zeta to membranes without affecting other PKC isoforms. This effect could be overcome by exogenous addition of arachidonic acid, suggesting that PKC-zeta may operate downstream of the activated PLA2. On the other hand, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, did not influence the amount of PKC-zeta associated with particulate fractions in control cells and could not abolish the PMA-mediated translocation of this isoform. Short-term exposure (45 min) of bone marrow cells to PMA, phorbol 12,13-dibutyrate or arachidonic acid increased the number of colonies formed over 7 days in a methylcellulose-based culture in vitro. The effects of PMA, but not those of arachidonic acid, could be prevented by putative PLA2 inhibitors. This suggests that PMA-mediated activation of conventional PKCs and novel PKCs leads to PLA2 activation which, by releasing arachidonic acid from phospholipids, activates PKC-zeta. This signalling pathway appears to be mitogenic for bone marrow cells.
Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1
Authors: Walker K N , Bottomley S P , Popplewell A G , Sutton B J , Gore M G
Published: August 2015

A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.
In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V
Authors: Dubois T , Oudinet J P , Russo-Marie F , Rothhut B
Published: August 2015

In order to understand how signal transduction occurs during T cell activation, it is necessary to identify the key regulatory molecules whose function is influenced by phosphorylation. Annexins II (A-II) and V (A-V) belong to a large family of Ca(2+)-dependent phospholipid-binding proteins. Among many putative functions, annexins may be involved in signal transduction during cellular proliferation and differentiation. In the present study we show that A-II is phosphorylated in vivo in the Jurkat human T cell line. Indeed, A-II is phosphorylated after stimulation by phorbol myristate acetate and on serine residues after T cell antigen receptor (TcR) stimulation. In cytosol from Jurkat cells, A-II is phosphorylated only by Ca2+/phospholipid-stimulated kinases such as Ca(2+)-dependent protein kinases C (cPKCs). A-V inhibits the phosphorylation of A-II and other substrates of cPKCs and has no effect on kinases activated only by phospholipids. In conclusion, A-II is phosphorylated both in vitro and in vivo in Jurkat cells, and may play a role as a substrate during signal transduction in lymphocytes via the TcR through the PKC pathway. On the other hand, A-V could act as a potent modulator of cPKCs in Jurkat cells.
Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids
Authors: Fosang A J , Last K , Gardiner P , Jackson D C , Brown L
Published: August 2015

We have developed a monoclonal antibody AF-28 that specifically recognizes a neo-epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...).
Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method
Authors: Dawson A P , Rich G T , Loomis-Husselbee J W
Published: August 2015

The rate of unidirectional efflux of 45Ca from rat liver microsomal vesicles loaded with 45Ca and then treated with thapsigargin is not inhibited by increased [Ca2+] in the external medium, although the net efflux rate is substantially inhibited. We have used this property to measure the electrochemical gradient of Ca2+ from the inside to the outside of the vesicles at a series of Ca2+ loadings, by measuring the external [Ca2+]free at which there is zero net efflux. At a loading of 7.9 +/- 0.6 nmol/mg of microsomal protein, the apparent internal [Ca2+]free is 21 +/- 1.6 microM. As the loading is increased, the internal [Ca2+]free increases linearly up to a value of 47 +/- 3.6 microM at a loading of 21.6 +/- 1.6 nmol/mg. Using a similar technique, the value for [Ca2+]free in the endoplasmic reticulum of permeabilized L1210 cells was found to be 12.5 microM.
Synergistic transcriptional activation of the thyrotropin receptor promoter by cyclic AMP-responsive-element-binding protein and thyroid transcription factor 1
Authors: Saiardi A , Falasca P , Civitareale D
Published: August 2015

In this study we have investigated the molecular mechanisms involved in hormonal induction of thyroid-specific transcription of the thyrotropin receptor (TSHr). A cyclic AMP-responsive element (CRE) has been characterized in the minimal TSHr promoter, and promoter activity shown to be also induced by thyroid transcription factor 1 (TTF-1). We here describe a cooperative effect between TTF-1 and CRE-binding protein on the TSHr promoter. Moreover we have identified a second TTF-1-binding site in the minimal promoter, which does not activate TSHr promoter activity but is required for the co-operative activation of the promoter. This report describes a new aspect of thyroid-specific gene expression, namely, how a generic extracellular signal can be interpreted in a thyroid-specific way.
Characterization of the mannose 6-phosphate-dependent pathway of lysosomal enzyme routing in an invertebrate
Authors: Alvarez V , Parodi A J , Couso R
Published: August 2015

The mannose 6-phosphate (Man6P)-dependent pathway for routing lysosomal enzymes was characterized in the hepatopancreas of the estuary crab Chasmagnatus granulata: (a) an acid alpha-L-fucosidase was purified to homogeneity from the above-mentioned organ and was shown to contain mannose-linked phosphate residues; (b) high-mannose-type oligosaccharides isolated from a protein fraction enriched in acid hydrolases were found to contain acid-labile N-acetylglucosamine (GlcNAc) residues; (c) a membrane-bound UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase was detected that phosphorylated the estuary-crab alpha-L-fucosidase and bovine uteroferrin but not bovine pancreas ribonuclease B; (d) a GlcNAc-1-phosphodiester alpha-N-acetylglucosaminidase that released GlcNAc units from GlcNAc alpha 1-P6Man alpha 1-methyl was detected in microsomal membranes of the hepatopancreas; (e) two detergent-solubilized microsomal proteins having molecular masses of 205 and 215 kDa that were retained by a Man6P-rich mannan-Sepharose column, from where they were eluted with Man6P but not with glucose 6-phosphate, were recognized by antisera raised against bovine large (215 kDa) and small (46 kDa) Man6P receptors. This is the first description of all the components of the Man6P-dependent mechanism for routing lysosomal enzymes in an invertebrate.
Some effects of post-translational N-terminal acetylation of the human embryonic ζ globin protein
Authors: Scheepens A , Mould R , Hofmann O , Brittain T
Published: August 2015

Using site-directed mutagenesis we have produced the first mutant form of a human embryonic haemoglobin. We have mutated the N-terminal Ser residue of the zeta-chain of haemoglobin Portland, zeta 2 gamma 2, (which is normally acetylated) to a Val (which possesses a free amine terminus). The protein spontaneously assembles into a fully functional tetramer which shows cooperative oxygen binding. Determination of the reactivity of the mutant protein with 2,3-diphosphoglycerate indicates that the mutation process does not lead to any major disruption of the protein structure. A comparison of the properties of the mutant and wild-type proteins identifies a significant role for the normal N-terminal acetylation of the zeta-chain with regard to the alkaline Bohr effect and the sensitivity of the oxygen affinity of the protein towards chloride ions. The possible physiological significance of this modification is discussed.
Cytosolic free Ca2+ oscillations induced by diadenosine 5′,5′′′-P1,P3-triphosphate and diadenosine 5′,5′′′-P1,P4-tetraphosphate in single rat hepatocytes are indistinguishable from those induced by ADP and ATP respectively
Authors: Green A K , Cobbold P H , Dixon C J
Published: August 2015

Diadenosine 5′,5‴-P1,P3-triphosphate (Ap3A) and diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) induce distinctive patterns of [Ca2+]i oscillations in single rat hepatocytes. We show here that [Ca2+]i oscillations induced by Ap3A and ADP are indistinguishable and that [Ca2+]i oscillations induced by Ap4A closely resemble those induced by ATP. These similarities embrace the following: (1) ADP and Ap3A invariably induce [Ca2+]i transients of short duration (approx. 9 s). Ap4A, like ATP, can induce, depending upon the individual cell, either transients of short duration (approx. 9 s), transients of much longer duration or a mixture of short and long transients within a single response. We show here that the pattern of oscillations induced by Ap4A is similar to that induced by ATP in the same hepatocyte. (2) Elevated intracellular cyclic AMP concentration modulates Ap3A-induced transients, like ADP-induced transients, through an increase in both the peak [Ca2+]i and the frequency of the transients. In contrast, Ap4A-induced transients, like ATP-induced transients, develop an increased duration or a sustained rise in [Ca2+]i, with no rise in peak [Ca2+]i. (3) Ap3A-induced transients, like ADP-induced transients, are abolished by low concentrations of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB; 5-10 nM), whereas long Ap4A-induced transients, like long ATP-induced transients, are refractory to high concentrations of PDB (100 nM). We propose that the [Ca2+]i oscillations induced in rat hepatocytes by Ap3A are mediated by the same purinoceptor that mediates the effects of ADP, whereas the oscillations induced by Ap4A are mediated by the same purinoceptor(s) that mediate the effects of ATP.
Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I
Authors: Teruel T , Valverde A M , Alvarez A , Benito M , Lorenzo M
Published: August 2015

Rat brown adipocytes at day 22 of foetal development showed greater size, higher mitochondria content and larger amounts of lipids, as determined by flow cytometry, than 20-day foetal cells. Simultaneously, an inhibition on the percentage of brown adipocytes into S+G2/M phases of the cell cycle was observed between days 20 and 22 of foetal development. The expression of several adipogenesis-related genes, such as fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase and insulin-regulated glucose transporter, increased at the end of foetal life in brown adipose tissue. In addition, the lipogenic enzyme activities and the lipogenic flux increased during late foetal development, resulting in mature brown adipocytes showing a multilocular fat droplet phenotype. Concurrently, brown adipocytes induced the expression of the uncoupling protein (UP) mRNA and UP protein, as visualized by immunofluorescence. The three isoforms of CCAAT enhancer-binding proteins (C/EBPs) were expressed at the mRNA level in brown adipose tissue at day 20. C/EBP alpha decreased and C/EBP beta and delta increased their expression between days 20 and 22 of foetal development, respectively. Brown adipose tissue constitutively expressed insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNAs. Moreover, IGF-IR mRNA content increased between days 20 and 22 in parallel with the occurrence of tissue differentiation.
Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I
Authors: Park E A , Mynatt R L , Cook G A , Kashfi K
Published: August 2015

The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene.
Differential expression of human Fas mRNA species upon peripheral blood mononuclear cell activation
Authors: Liu C , Cheng J , Mountz J D
Published: August 2015

Human Fas/Apo-1 is a cell-surface protein that mediates apoptosis upon ligation with Fas ligand. The gene lies on the long arm of chromosome 10, consists of nine exons, and spans more than 26 kb of DNA. We previously reported the presence of a Fas variant mRNA, designated as Fas delta TM, in human peripheral blood mononuclear cells. Fas delta TM is generated by alternative splicing of the intact exon 6, which encodes the Fas transmembrane domain. In the present study, we describe three novel forms of Fas mRNA that are generated by alternative splicing of exons 3, 4, 6 and 7. These three mRNA variants undergo a frameshift and produce truncated polypeptides because of the appearance of a stop codon in the altered open reading frame. On activation of the peripheral blood mononuclear cells, a decreased expression of alternatively spliced Fas mRNA species correlated with increased cell-surface expression of Fas. These results suggest that differential expression of alternatively spliced Fas mRNAs may play a role in regulation of Fas function via regulation of the production of the membrane-bound and the soluble, secreted Fas protein products.
Biochemical changes in the collagenous matrix of osteoporotic avian bone
Authors: Knott L , Whitehead C C , Fleming R H , Bailey A J
Published: August 2015

No detailed biochemical analysis has been carried out of the compositional changes in the collagen matrix of avian bone in relation to increased bone fragility in osteoporosis. We have shown that osteoporosis in avian bone is certainly not just a simple loss of apatite and collagen, but involves significant changes in the biochemistry of the collagen molecule and consequently in the physical properties of the fibre. The decreased mechanical strength and the change in the thermal stability can be directly related to changes in post-translational modifications, i.e. lysine hydroxylation and the intermolecular cross-link profile. The increased hydroxylation and change in cross-linking are consistent with increased turnover of the collagen, possibly in an attempt to initiate a repair mechanism which, in fact, leads to an acceleration in the increase in fragility of the bone. Clearly there are post-translational modifications of the newly synthesized collagen in avian osteoporosis, and these changes may play a role in the pathogenesis of the disease.
Insulin and insulin-like growth factor I (IGF-I) stimulate GLUT4 glucose transporter translocation in Xenopus oocytes
Authors: Mora S , Kaliman P , Chillarón J , Testar X , Palacín M , Zorzano A
Published: August 2015

1. The heterologous expression of glucose transporters GLUT4 and GLUT1 in Xenopus oocytes has been shown to cause a differential targeting of these glucose-carrier isoforms to cellular membranes and a distinct induction of glucose transport activity. In this study we have evaluated the effect of insulin and insulin-like growth factor I (IGF-I) on glucose uptake and glucose transporter distribution in Xenopus oocytes expressing mammalian GLUT4 and GLUT1 glucose carriers. 2. Insulin and IGF-I stimulated 2-deoxyglucose uptake in GLUT4-expressing oocytes, but not in GLUT1-expressing oocytes or in water-injected oocytes. The stimulatory effect of insulin and IGF-I on 2-deoxyglucose uptake in GLUT4-expressing oocytes occurred via activation of the IGF-I receptor. 3. Subcellular-fractionation studies indicated that insulin and IGF-I stimulated translocation of GLUT4 to the cell surface of the oocyte. 4. Incubation of intact oocytes with insulin stimulated phosphatidylinositol 3-kinase activity, an effect that was blocked by the additional presence of wortmannin. Furthermore, wortmannin totally abolished the insulin-induced stimulation of 2-deoxyglucose uptake in GLUT4-expressing oocytes. 5. In this study, both the insulin-induced GLUT4 carrier translocation and GLUT4-dependent insulin-stimulated glucose transport have been reconstituted in the Xenopus oocyte. These observations, together with the fact that wortmannin, as found in adipocytes, inhibits insulin-stimulated glucose transport in oocytes, suggest that the heterologous expression of GLUT4 in oocytes is a useful experimental model by which to study the cell biology of insulin-induced GLUT4 translocation.
Quantification of signalling components and amplification in the β-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes
Authors: Post S R , Hilal-Dandan R , Urasawa K , Brunton L L , Insel P A
Published: August 2015

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5′-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.
Identification and subcellular localization of sphinganine-phosphatases in rat liver
Authors: De Ceuster P , Mannaerts G P , Van Veldhoven P P
Published: August 2015

One of the primary products of [4,5-3H]sphinganine phosphate, added to fibroblast cultures, is sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.
Extracellular ATP activates different signalling pathways in rat Sertoli cells
Authors: Foresta C , Rossato M , Bordon P , Di Virgilio F
Published: August 2015

1. The present study describes effects of extracellular ATP (ATPe) on plasma membrane potential and cytoplasmic Ca2+ concentrations ([Ca2+]i) in rat Sertoli cells. Sertoli cells in suspension were stimulated with ATPe and other nucleotides and ionic changes were monitored utilizing the fluorescent dyes bis-oxonol and fura-2/AM. ATPe induced a prompt plasma membrane depolarization which was dependent on Na+ influx from the extracellular medium, since it was abolished by omission of extracellular Na+. Depolarization was independent of [Ca2+]i rise as it also occurred in the absence of extracellular Ca2+ and after intracellular Ca2+ stores were discharged with thapsigargin. ATPe also stimulated a rapid and biphasic increase in [Ca2+]i: a prompt spike was followed by a prolonged sustained plateau. The initial spike was dependent on Ca2+ release from intracellular stores since it was also present when cells were incubated in EGTA-supplemented Ca(2+)-free medium and was abolished by pretreatment with ionomycin and thapsigargin, agents that discharge intracellular Ca2+ stores. The sustained phase was dependent on Ca2+ influx from the extracellular medium as it was abolished when cells were incubated in EGTA-supplemented Ca(2+)-free medium. Ca2+ influx was due to activation of voltage-operated calcium channels (VOCCs) since it was abolished by the VOCC inhibitors verapamil and nifedipine or incubation in sucrose medium, an experimental condition which precludes plasma membrane depolarization by ATPe. 2. ATPe-induced rises in intracellular Ca2+ concentration and plasma membrane depolarization were reduced by pretreatment with pertussis toxin, suggesting that ATPe-activated transduction mechanisms are in part under the control of pertussis toxin-sensitive G-proteins. These data show that Sertoli cells possess P2-purinergic receptor subtypes coupled to influx of Na+ and release of Ca2+ from intracellular stores and provide evidence for an activation of different pathways by extracellular ATPe. Activation of these receptors induces Na+ influx that causes a rapid plasma membrane depolarization. Furthermore, ATPe also triggers Ca2+ release from intracellular stores and Ca2+ influx from extracellular space via dihydropyridine-sensitive VOCCs.
Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases
Authors: Pol E , Olsson S L , Estrada S , Prasthofer T W , Björk I
Published: August 2015

The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the ‘occluding loop’ of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.
Lysosomal processing of amyloid precursor protein to Aβ peptides: a distinct role for cathepsin S
Authors: Munger J S , Haass C , Lemere C A , Shi G P , Wong W S F , Teplow D B , Selkoe D J , Chapman H A
Published: August 2015

To investigate the potential contribution of the lysosomal compartment in the processing of amyloid precursor protein (APP) to amyloid beta-peptides (A beta s), we stably overexpressed a series of lysosomal proteases (the cysteine proteases, cathepsins B, L and S, and the aspartic protease, cathepsin D) in a human kidney epithelial cell line (293) transfected to express high levels of beta APP. Preliminary experiments indicated that 293 cells endogenously synthesize cathepsins B, L and D, but not cathepsin S. A beta secretion was assessed by immunoprecipitation and ELISA and found to be increased approximately 2-fold following cathepsin S expression, but to be unchanged (cathepsins B, L) or decreased (cathepsin D) in the other double transfectants. E-64d, an inhibitor of lysosomal cysteine proteases, significantly reduced A beta secretion by the cathepsin S transfectants, but had no effect on cells expressing the other proteases. Radiosequencing of A beta secreted by cathepsin S-expressing cells revealed that a previously unreported variant beginning at Met -1 (relative to the most common A beta N-terminus, Asp -1) accounted for most of the increase in A beta secretion. Immunostaining of human brain sections revealed cathepsin S in cortical neurons and glia in samples of brain from patients with Alzheimer's disease. These results provide evidence in living cells for a pathway in which cathepsin S generates A beta from amyloidogenic fragments of beta APP in the endosomal/lysosomal compartment. This pathway appears to be inducible, distinct from a constitutive pathway used by 293 and other cells to generate A beta, and may be relevant to the pathogenesis of Alzheimer's disease.
Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two non-steroidal anti-inflammatory drugs: tolmetin and zomepirac
Authors: Zia-Amirhosseini P , Ding A , Burlingame A L , McDonagh A F , Benet L Z
Published: August 2015

Human serum albumins modified by covalently bound tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS. Three binding sites were common to both products, i.e. lysine-195, -199 and -351. Three further modified residues were identified for the tolmetin-albumin product, i.e. aspartic acid 1, and lysine-524 and -536.
Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fc γ-IIA receptor
Authors: Yanaga F , Poole A , Asselin J , Blake R , Schieven G L , Clark E A , Law C L , Watson S P
Published: August 2015

Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fc gamma-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fc gamma-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fc gamma-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fc gamma-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.
Pretranslational regulation of pyruvate dehydrogenase complex subunits in white adipose tissue during the suckling-weaning transition in the rat
Authors: Maury J , Kerbey A L , Priestman D A , Patel M S , Girard J , Ferre P
Published: August 2015

Total pyruvate dehydrogenase complex activity is low in white adipose tissue during the suckling period and increases markedly at weaning on to a high-carbohydrate diet. This is concomitant with an increase in the E1 alpha, E1 beta and E2 subunit protein concentration and their respective mRNAs, suggesting a pretranslational control of this phenomenon. The most marked change is seen for the E1 alpha subunit (17-fold increase in protein concentration). The changes in pyruvate dehydrogenase complex activity and subunit abundance induced by weaning on to a high-carbohydrate diet are precluded if the animals are weaned on to a high-fat diet, suggesting that the nutritional and/or related hormonal changes rather than a developmental stage are responsible for the observed adipose-tissue pyruvate dehydrogenase complex pattern.
Insulin stimulation of intracellular free Ca2+ recovery and Ca2+-ATPase gene expression in cultured vascular smooth-muscle cells: role of glucose 6-phosphate
Authors: Kim Y C , Zemel M B
Published: August 2015

Wa have previously reported that insulin accelerates recovery of intracellular Ca2+ concentrations ([Ca2+]i) from pressor agonist-induced Ca2+ loads and stimulates both plasmalemmal and sarcoplasmic-reticulum Ca(2+)-ATPase gene expression in cultured and freshly isolated vascular smooth-muscle cells (VSMCs), suggesting that insulin attenuation of vascular tone may result from modulation of [Ca2+]i. Accordingly, we have now evaluated the linkage between this insulin-regulation of VSMC[Ca2+]i and classical actions of insulin (i.e. glucose transport and metabolism). Cultured VSMCs were incubated in the presence or absence of insulin in a medium containing either pyruvate, glucose, 3-O-methylglucose or 2-deoxyglycose. Insulin caused an 87% increase in [Ca2+]i recovery rate after stimulation with arginine-vasopressin (P < 0.01) and caused a marked increase in Ca(2+)-ATPase mRNA and protein levels in the presence of glucose. Comparable increases in both [Ca2+]i recovery and Ca2(+)-ATPase expression were found when glucose was replaced by 2-deoxyglucose. In contrast, no stimulation was found in either the glucose-free or 3-O-methylglucose-containing medium. As both glucose analogues are transported, but only 2-deoxyglucose is phosphorylated, this indicates that glucose transport and metabolism to glucose 6-phosphate is essential for insulin regulation of VSMC [Ca2+]i, possibly via a glucose-6-phosphate-dependent carbohydrate-response element in the Ca2(+)-ATPase gene.
Role of cyclic GMP in the control of capacitative Ca2+ entry in rat pancreatic acinar cells
Authors: Gilon P , Obie J F , Bian X , Bird G S J , Putney J W
Published: August 2015

We have investigated the possible roles of cyclic GMP (cGMP) in initiating or regulating capacitiative Ca2+ entry in rat pancreatic acinar cells. In medium containing 1.8 mM external Ca2+, thapsigargin activated Ca2+ entry and slightly but significantly increased intracellular cGMP concentration. This rise in cGMP levels was prevented by pretreating the cells with the guanylate cyclase inhibitor, LY-83583, or by omitting Ca2+ during stimulation by thapsigargin or methacholine. LY-83583 and NG-nitro-L-arginine (L-NA, an inhibitor of NO synthase) both had a small inhibitory effect on Ca2+ entry when they were added after thapsigargin in Ca2(+)-containing medium, and they reduced by 32 and 48% respectively the thapsigargin-induced capacitative Ca2+ entry when added to the cells during a 20 min preincubation period. However, neither dibutyryl cGMP (Bt2cGMP) nor sodium nitroprusside, an NO mimic, affected either basal intracellular Ca2+ concentration [Ca2+]i or thapsigargin-induced capacitative Ca2+ entry. Further, the inhibitory effects observed after preincubation with LY-83583 or L-NA could not be prevented by preincubation with Bt2cGMP, nor could they be reversed by adding Bt2cGMP, 8-bromo-cGMP or sodium nitroprusside acutely after activation of capacitative Ca2+ entry by thapsigargin. Finally, pretreatment of cells with LY-83583 or L-NA did not affect Ca2+ signalling in response to 1 microM methacholine, including the pattern of [Ca2+]i oscillations. In conclusion, in pancreatic acinar cells, the rise in cellular cGMP levels appears to depend on, rather than cause, the increase in [Ca2+]i with agonist stimulation.