bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes
Authors: PLÉE-GAUTIER Emmanuelle , GROBER Jacques , DUPLUS Eric , LANGIN Dominique , FOREST Claude
Published: August 2015

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 µM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 µM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.
Maintenance of cellular levels of G-proteins: different efficiencies of αs and αo synthesis in GH3 cells
Authors: LI Ying , MENDE Ulrike , LEWIS Carol , NEER Eva J
Published: August 2015

G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein α, β and γ subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. We have assayed the steady-state levels of mRNA and protein for two types of G-protein subunits, αs and αo, in rat brain, heart and GH3 cells, and found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of αs protein than to produce the same amount of αo protein. Such a situation could arise from a relatively rapid rate of αs protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of αs mRNA compared with αo mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for αs than for αo, yet the rate of αs protein synthesis is only 9 times greater than αo protein synthesis. The degradation rates of the two proteins are similar (13 h for αs and 18 h for αo). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of αs than αo, we asked whether there is a pool of αs mRNA that does not participate in protein synthesis. We found that virtually all αs and αo mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, our results suggest that the relatively slow rate of αs protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination.
Association of insulin-degrading enzyme with a 70 kDa cytosolic protein in hepatoma cells
Authors: AUTHIER François , CAMERON Pamela H. , TAUPIN Véronique
Published: August 2015

We have investigated the biosynthesis, subcellular location and expression of insulin-degrading enzyme (IDE), a type-I peroxisomal protease, in semi-permeabilized hepatoma cells using pulse-chase experiments, non-denaturing immunoprecipitation protocols and Northern-blot analyses. In HepG2 cell lysates prepared from cells radiolabelled with Tran[35S]-label, immunoprecipitated IDE was observed immediately after a 5 min pulse and subsequently declined during chase with t½ of approx. 33 h. In addition to the 110 kDa IDE protein, a protein of 70 kDa (p70) was identified in radiolabelled immunoprecipitates when using a monoclonal anti-IDE antibody 9B12 under non-denaturing conditions. This same antibody did not recognize p70 on Western blots of whole-cell lysates nor in sequential immunoprecipitates of immunocomplex-bead eluates from anti-IDE immunoprecipitations. Likewise, cross-linking studies performed on intact HepG2 and H35 hepatoma cells in vivo revealed the existence of a hetero-oligomeric complex of 180 kDa in which IDE and p70 were physically associated. Digitonin-permeabilization studies in normal and 35S-labelled HepG2 cells have defined a predominant association of IDE and its associated protein p70 with cytosol (supernatant); only a minor amount of the protein IDE was detected in peroxisomes (cellular pellet). Immunoprecipitation of IDE from 35S-labelled cell lysates of normal and stably transfected Chinese hamster ovary cells overexpressing IDE failed to detect p70. Treatment of HepG2 cells with clofibrate, a peroxisome proliferator, resulted in a dose-dependent increase of the two human IDE transcripts of 3.6 and 3.2 kb. This effect was not accompanied by a similar change at the protein level, nor by a change in the subcellular location of the proteins IDE and p70. Based on these findings we propose that in hepatoma cells: (1) IDE mainly exists in a stable cytoplasmic pool that is unchanged in cells undergoing peroxisomal proliferation; and (2) p70 binding to IDE may serve to maintain the dual cytosolic and peroxisomal pools of IDE in a stable equilibrium.

Receieved 11 March 1996/30 May 1996; accepted 25 June 1996
Transfection of human topoisomerase IIα into etoposide-resistant cells: transient increase in sensitivity followed by down-regulation of the endogenous gene
Authors: ASANO Takeshi , AN Taeha , MAYES Janice , ZWELLING Leonard A. , KLEINERMAN Eugenie S.
Published: August 2015

We have investigated the possibility of overcoming the resistance of human brain tumour cells (HBT20) to etoposide by transferring the normal human topoisomerase IIα (H-topo II) gene into these cells. H-topo II in a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumour virus (MMTV) promoter was transfected into etoposide-resistant HBT20 cells (HBT20-hTOP2MAM). HBT20 cells transfected with pMAMneo vector alone served as control cells (HBT20-MAM). These were stable transfections. Following a 2 h dexamethasone treatment, H-topo II mRNA expression, protein production, etoposide-induced DNA-protein complex formation and sensitivity to etoposide were increased in HBT20-hTOP2MAM cells compared with control HBT20-MAM cells and with HBT20-hTOP2MAM cells not treated with dexamethasone. However, mRNA and protein levels and cell sensitivity returned to baseline when incubation with dexamethasone was continued for 24 h. This decrease from the 2 h values could not be explained by a loss of the MMTV promoter response to dexamethasone. (H-topo IIα promoter)-(chloramphenicol acetyltransferase) constructs containing regions -559–0 and -2400–0 were significantly down-regulated in HBT20-hTOP2MAM cells treated for 24 h with dexamethasone compared with dexamethasone-treated control cells. H-topo II mRNA stability after 24 h of dexamethasone treatment was not altered compared with that in control cells. Our data indicate that the exogenously produced H-topo II may have a negative-feedback effect on the endogenous topoisomerase II promoter, causing down-regulation of the endogenous gene.
Domain-structure analysis of recombinant rat hormone-sensitive lipase
Authors: ØSTERLUND Torben , DANIELSSON Birgitta , DEGERMAN Eva , CONTRERAS Juan Antonio , EDGREN Gudrun , DAVIS Richard C. , SCHOTZ Michael C. , HOLM Cecilia
Published: August 2015

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the α/β-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and α/β-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.
Expression of the human spermidine/spermine N1-acetyltransferase in spermidine acetylation-deficient Escherichia coli
Authors: IGNATENKO Natalia A , FISH Jennifer L , Richard SHASSETZ L. , WOOLRIDGE Dale P , GERNER Eugene W
Published: August 2015

A cDNA encoding the human spermidine/spermine N1-acetyltransferase (N1SSAT) was conditionally expressed in a strain of Escherichia coli deficient in spermidine-acetylating activity. Conditional expression of this cDNA was performed under the control of the lac promoter, by addition of the non-hydrolysable lactose analogue isopropyl β-D-thiogalactoside. Expression of the N1SSAT cDNA oriented in the sense direction resulted in the acetylation of spermidine at the N1 but not the N8 position and a decrease in endogenous spermidine contents and growth rates in these bacteria. When this cDNA was expressed in the anti-sense orientation, spermidine acetylation was not detected and endogenous spermidine contents and growth rates were unaffected. Increasing the endogenous N1-acetylspermidine concentration by addition of this amine to the culture medium did not suppress growth, and increasing endogenous spermidine pools by exogenous addition was not sufficient to restore optimal growth in cells expressing the human N1SSAT. Exogenous spermidine, but neither N1- nor N8-acetylspermidine, stimulated cell growth in strains unable to synthesize spermidine. These results suggest that one physiological consequence of spermidine acetylation in E. coli is growth inhibition. The mechanism of this inhibition seems to involve the formation of acetylspermidine, and is not simply due to a decrease in the intracellular concentration of non-acetylated spermidine.
Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigor-contracture of metabolically compromised single cardiomyocytes
Authors: ALLUE Isidro , GANDELMAN Olga , DEMENTIEVA Ekaterina , UGAROVA Natalya , COBBOLD Peter
Published: August 2015

Cytoplasmic ATP can be measured continuously in single cardiac myocytes by monitoring the luminescence from microinjected firefly luciferase. We show here that the signals are markedly influenced by changes in cytoplasmic pH, and the calibration of the signals as ATP concentration is markedly affected by cytoplasmic protein. Measurements with a pH-sensitive fluorescent dye show that intracellular pH (pHi) can be clamped at pH 7.08 by perfusing cells with a modified bicarbonate-buffered Krebs saline containing 92 mM NaHCO3 and equilibrated with 20% CO2. Calibration of the firefly luciferase signal in vitro in the presence of high concentrations of BSA (180 mg/ml), to simulate the cytoplasmic protein concentration, revealed a shift in Km (ATP) to 2 mM, from approx. 400 µM in the absence of albumin in an identical ionic milieu. Luciferase measurements in pH-clamped cells show that metabolically poisoned isolated rat ventricle cardiomyocytes enter rigor at a cytoplasmic ATP concentration of between 1 and 2 mM. As the cells shorten in rigor, a process that is complete in 30–40 s, the cytoplasmic ATP concentration falls simultaneously to a level of typically 20 µM. When cyanide is removed 10 min later, to simulate reoxygenation, the signal recovers over a period of 2–3 min to a level approx. 70% of the original in the healthy cell. These studies indicate that rigor-mediated depletion of cytoplasmic ATP in metabolically poisoned cardiomyocytes is considerably more extreme than hitherto indicated.
Arachidonic acid activates a proton conductance pathway and the Na+/H+ exchanger in platelets
Authors: CAVALLINI Lucia , COASSIN Mariagrazia , BOREAN Alessio , ALEXANDRE Adolfo
Published: August 2015

The treatment of aspirinated platelets with the endomembrane Ca2+-ATPase inhibitor thapsigargin (Tg) induces a large increase in cytosolic pH (pHi), as measured with the intracellular fluorescent indicator 2´,7´-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. In contrast, Tg induces a decrease in pHi in the presence of the Na+/H+ exchanger inhibitor 5-(N,N-hexamethylene)-amiloride (NHA). Both effects are inhibited if the cytosolic free Ca2+ concentration ([Ca2+]i) is chelated by loading with bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra-acetoxymethyl ester (BAPTA-AM). Without BAPTA, the pH effects are inhibited in the presence of BSA or the phospholipase A2 inhibitor oleoyloxyethylphosphocholine. These observations are consistent with the Tg-induced pH effects being mediated at least in part by the release of arachidonic acid (ArA) on activation of phospholipase A2 by the increased [Ca2+]i. Exogenous ArA promotes a rapid decrease in pHi in platelets suspended in a high-[Na+] medium, and an increase in pHi if platelets are depolarized by suspension in a high-[K+] medium in the presence of valinomycin and the external pH is increased to 7.9. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) behaves like ArA, although ArA is not a protonophore. It is concluded that ArA activates a proton conductance across the plasma membrane. The latter is inhibited by La3+. In high-[Na+] media, the pHi previously decreased by ArA recovers rapidly on removal of ArA with BSA. The effect is prevented by NHA. The recovery after BSA is much slower if FCCP rather than ArA is used to decrease pHi, but it is fast again with both ArA and FCCP. Furthermore, pHi previously decreased by ArA also recovers readily on inhibition of the ArA-activated H+ conductance with La3+, and the effect is NHA-sensitive. When pHi is decreased with the K+/H+ ionophore nigericin, a rapid recovery is activated by ArA followed by BSA (but not by BSA alone). The effect is independent of Ca2+ and protein kinase C. It is concluded that ArA, besides activating the H+ conductance, also acts as an activator of the Na+/H+ exchanger.
Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes
Authors: RENAUD Jean-Paul , DAVYDOV Dmitri R. , HEIRWEGH Karel P. M. , MANSUY Daniel , HOA Gaston HUI BON
Published: August 2015

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177–185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.
Biochemical alterations in collagen IV induced by in vitro glycation
Authors: RAABE Hans-M. , MOLSEN Hilke , MLINARIC Snjezana-M. , AÇIL Yahya , SINNECKER Gernot H. G. , NOTBOHM Holger , KRUSE Klaus , MÜLLER Peter K.
Published: August 2015

Non-enzymic interactions of carbohydrates and proteins are a major feature of cumulative modification in basement membranes in the course of diabetic microvascular complications. To evaluate the significance of both glycation and glycoxidation reactions for subsequent alterations of biochemical properties, we examined the effects of in vitro glycation on distinct collagen IV domains under different experimental conditions. The 7 S domain and the major triple-helical domain from human placental collagen IV were incubated for various time intervals up to 14 days at 37 °C in the presence of different concentrations of either glucose or ribose under oxidative and antioxidative conditions. Carbohydrate-induced non-enzymic modification in two collagen IV domains was revealed by increased cross-linking and fluorescence. In addition, these non-enzymic modifications apparently have a major impact on molecular conformation and thermal stability of collagen IV, which in turn might influence both cell-matrix interactions and matrix assembly.
Multiple factors regulating the expression of human thromboxane synthase gene
Authors: LEE Kuan-Der , BAEK Seung Joon , SHEN Rong-Fong
Published: August 2015

Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, ∼75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (∼55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.
The lysosomal Ca2+ pool in MDCK cells can be released by ins(1,4,5)P3-dependent hormones or thapsigargin but does not activate store-operated Ca2+ entry
Authors: HALLER Thomas , VÖLKL Harald , DEETJEN Peter , DIETL Paul
Published: August 2015

In several cell types, Ca2+ release from intracellular Ca2+ stores by Ins(1,4,5)P3 elicits Ca2+ influx from the extracellular space into the cytoplasm, termed store-operated Ca2+ entry (SOCE). In MDCK cells, the Ins(1,4,5)P3-sensitive Ca2+ store giving rise to SOCE essentially overlaps with the thapsigargin (TG)-sensitive store. Recent evidence suggests that in MDCK cells lysosomes form a Ca2+ pool that is functionally coupled with the Ins(1,4,5)P3-sensitive Ca2+ store: Ca2+ can be selectively released from lysosomes by glycyl-L-phenylalanine naphthylamide, an agent inducing lysosomal swelling with subsequent and reversible permeabilization of the vesicular membranes. This compartment is also depleted by Ins(1,4,5)P3-dependent agonists or TG, indicating that it is part of a larger, Ins(1,4,5)P3-sensitive Ca2+ pool. Here we show that whereas SOCE is triggered by Ca2+ release from the entire Ins(1,4,5)P3-sensitive Ca2+ pool, selective Ca2+ release from lysosomes alone is unable to trigger SOCE. This finding is consistent with measurements of the store-operated cation current, a direct parameter for store-operated Ca2+ and Na+ entry into MDCK cells. Hence it is proposed that the Ins(1,4,5)P3-sensitive Ca2+ pool is composed of different intracellular compartments that do not uniformly stimulate Ca2+ entry into the cell.
Inhibition patterns of a model complex mimicking the reductive half-reaction of sulphite oxidase
Authors: CHAUDHURY Pradeep K , DAS Samar K , SARKAR Sabyasachi
Published: August 2015

Different inhibition types of the saturation kinetics involving a synthesized model complex, [Bu4N]2[MoVIO2(mnt)2] (E) (where mnt2- = 1,2-dicyanoethylenedithiolate), and HSO3- as the substrate (S) by structurally similar anions SO42-, H2PO4- and H2PO3- have been shown for the first time in relevance to the reductive half reaction of the native enzyme sulphite oxidase. SO42- acts as a competitive inhibitor. The mixed-type non-competitive inhibition by H2PO4- and the sigmoidal-type inhibition by H2 PO3- are explained by a diamond-configuration random-order model. This involves a random binding sequence of the substrate and the inhibitor, and forms, in addition to two binary complexes [enzyme-substrate (ES) and enzyme-inhibitor (EI)], one enzyme-substrate-inhibitor-type ternary complex (ESI) by participation of at least one more binding site in addition to the catalytic site. This is possible in the present case only by co-ordination enhancement of molybdenum in E. This co-ordination expansion is brought about by nucleophilic attack of the substrate or the inhibitor at the molybdenum, forming a hepta-coordinated binary complex with the generation of an oxoanionic functional site, called the allosteric site. Analysis of the experimental data suggests that the inhibition by H2PO4- is due to the mechanism following either equilibrium conditions or a combination of steady-state and equilibrium conditions. With H2PO3-, the inhibition is due to the mechanism following the steady-state conditions. It is also shown that the ternary complex involving the enzyme, substrate and H2PO4- or H2 PO3- is productive, but at a lower rate than that of the enzyme-substrate binary complex. Mixed-type inhibition with H2PO4- is actually of the type called ‘partially mixed competitive and non-competitive’ as the inhibitor binds both to the catalytic site and to the allosteric site. The sigmoidal-type inhibition by H2PO3- is similar to heterotropic allosteric effect of mixed V,K type with the distinction, however, that the significance of co-operativity in this case is of kinetic importance only.

Received 3 January 1996/20 May 1996; accepted 25 June 1996
Analysis of the interactions between an enzyme and multiple inhibitors using combination plots
Authors: ASANTE-APPIAH Ernest , CHAN William W.-C.
Published: August 2015

The concurrent effects of two enzyme inhibitors have been analysed previously with the Yonetani-Theorell plot to obtain insight into the interactions between bound inhibitors. This procedure, like many other traditional graphical methods in enzymology, is based on the estimation of intersecting tendencies in a family of lines. In a recent paper from this laboratory [Chan (1995) Biochem. J. 311, 981–985] it was shown that a plot of this nature may sometimes be replaced, with advantage, by a ‘combination plot’ in which all data points are accommodated in a single line. We have now extended this approach to analyse the effects of multiple inhibitors and have developed combination plots which illustrate the interaction behaviour in an optimal manner. Thus, in these plots, the synergistic or antagonistic nature of the interactions is clearly evident from the slope, which also provides a direct estimate of the interaction coefficient. The analysis is more efficient and consequently requires fewer enzyme assays. This approach is applicable to various special cases, including that in which three inhibitors bind simultaneously to the enzyme.
Identification of an active site cysteine residue in human type I Ins(1,4,5)P3 5-phosphatase by chemical modification and site-directed mutagenesis
Authors: COMMUNI David , ERNEUX Christophe
Published: August 2015

Chemical modification using thiol-directed agents and site-directed mutagenesis have been used to investigate the crucial role of an active site cysteine residue within the substrate-binding domain of human type I Ins(1,4,5)P3 5-phosphatase. Irreversible inhibition of enzymic activity is provoked by chemical modification of the enzyme by N-ethylmaleimide (NEM), 5,5´-dithio-2-nitrobenzoic acid, iodoacetate and to a much smaller extent by iodoacetamide. The alkylation reaction by NEM is prevented in the presence of Ins(1,4,5)P3. The results indicate that NEM binds at the active site of the enzyme with a stoichiometry of 0.9 mol of NEM per mol of enzyme. A single [14C]NEM-modified peptide was isolated after α-chymotrypsin proteolysis of the radiolabelled enzyme and reverse-phase HPLC. Sequence analysis of the active site-labelled peptide (i.e. MNTRCPAWCD) demonstrated that Cys348 contained the radiolabel. Furthermore two mutant enzymes were obtained by site-directed mutagenesis of the cysteine residue to serine and alanine respectively. Both mutant enzymes had identical UV CD spectra. The two mutants (i.e. Cys348 → Ser and Cys348 → Ala) show a marked loss of enzymic activity (more than 98% compared with the wild-type enzyme). Thus we have directly identified a reactive cysteine residue as part of the active site, i.e. the substrate-binding domain, of Ins(1,4,5)P3 5-phosphatase. This cysteine residue is part of a sequence 10 amino acids long that is well conserved among the primary structures of inositol and phosphatidylinositol polyphosphate 5-phosphatases.
Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase
Authors: WERNER Ernst R. , PITTERS Eva , SCHMIDT Kurt , WACHTER Helmut , WERNER-FELMAYER Gabriele , MAYER Bernd
Published: August 2015

The binding of tetrahydropteridines with 6-di- and trihydroxypropyl side chains to recombinant rat neuronal nitric oxide (NO) synthase (EC 1.14.13.39) was determined by competition with 6R-[3´-3H]-5,6,7,8-tetrahydro-L-erythro-biopterin (6R-[3´-3H]H4biopterin). Although all but one of the derivatives exhibited only poor affinities (Ki 50 µM), the 4-amino analogue of 6R-H4biopterin was a potent antagonist of 6R-H4biopterin binding (Ki 13.2 nM). The 4-amino analogue of 6R-H4 biopterin inhibited NO synthase stimulation by the natural cofactor 6R-H4biopterin with an IC50 of 1 µM without affecting the basal activity observed in the absence of added 6R-H4biopterin. Because the 4-amino analogue of 6R-H4biopterin also inhibited dihydropteridine reductase (EC 1.6.99.7; IC50 20 µM), our results support the hypothesis that redox cycling of H4biopterin might be required for the NO synthase reaction.
A steady-state labelling approach to the measurement of proteoglycan turnover in vivo and its application to glomerular proteoglycans
Authors: AKUFFO Emma L , HUNT Jayne R , MOSS Jill , WOODROW David , DAVIES Malcolm , MASON Roger M.
Published: August 2015

Rats were implanted with mini osmotic pumps delivering sodium [35S]sulphate and their newly synthesized proteoglycans were labelled over a 146 h period (steady-state labelling). Proteoglycan turnover was measured in vivo using a chase protocol. Glomerular proteoglycans were recovered quantitatively and the perlecan present was isolated by immunoprecipitation. The procedure allows newly synthesized proteoglycans to be quantified in mass units (pmol of glycosaminoglycan sulphate) after labelling and during the chase. Ultrastructural-immunogold experiments identified the location of perlecan as the glomerular basement membrane and mesangial matrix. Perlecan in the basement membrane was quantified using the ultrastructural-immunogold technique. Perlecan comprises about 10% of the total glomerular proteoglycans, which are otherwise associated with glomerular cells and the mesangium. Both the total glomerular heparan sulphate proteoglycans and perlecan turn over rapidly (t½ ∼ 3–4 h and < 3 h respectively). In contrast, turnover of proteoglycans in other tissues was slow, except in the liver where the heparan sulphate and chondroitin sulphate t½ values were 16 h and 9 h respectively. Microalbuminuria was induced with a low-dose regimen of puromycin aminonucleoside. At the onset of microalbuminuria (5 days) there was no change in the level of newly synthesized perlecan, or in perlecan in the glomerular basement membrane detected by immunogold labelling. Newly synthesized perlecan had undergone a minimal change in turnover rate by day 5 in puromycin aminonucleoside-treated rats. In contrast, the total glomerular proteoglycan population showed a dramatic decrease in turnover by day 5. Since there was no evidence of accumulation of glomerular proteoglycans on either day 5 or day 6, it is likely that decreased turnover of cell-associated proteoglycans is accompanied by an equivalent decrease in their synthesis.
Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes
Authors: MINETTI Giampaolo , PICCININI Giampiero , BALDUINI Cesare , SEPPI Claudio , BROVELLI Augusta
Published: August 2015

Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca2+-activated K+ channel, and the only known inhibitor of Ca2+-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K+-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16–23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca2+-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required.
Locally formed 5-hydroxytryptamine stimulates phosphate transport in cultured opossum kidney cells and in rat kidney
Authors: HAFDI Zakia , COUETTE Sylvianne , COMOY Etienne , PRIE Dominique , AMIEL Claude , FRIEDLANDER Gerard
Published: August 2015

Renal proximal tubular cells have been shown to express aromatic l-amino acid decarboxylase (l-AAAD), which converts l-dopa into dopamine and 5-hydroxytryptophan [(OH)Trp] into 5-hydroxytryptamine (5-HT; serotonin). Because 5-HT receptors have been demonstrated in proximal cells, we hypothesized that 5-HT may act as an autocrine/paracrine modulator of proximal transport. We evaluated this possibility in opossum kidney (OK) cells, a renal epithelial cell line with a proximal phenotype expressing 5-HT1B receptors, and in intact anaesthetized rats. 5-HT synthesis by OK cells increased with incubation time and (OH)Trp concentration, and was abolished by benserazide, an l-AAAD inhibitor. 5-HT reversed parathyroid hormone (PTH)-induced cAMP accumulation in a pertussis toxin-sensitive manner and reduced the PTH inhibition of Pi uptake without affecting the NaPi-4 mRNA level. The effects of 5-HT on cAMP generation and Na–Pi co-transport were reproduced by (OH)Trp, except in the presence of benserazide, and by l-propranolol and dihydroergotamine, two 5-HT1B receptor agonists. In rats, (OH)Trp and dihydroergotamine decreased fractional Pi excretion. Benserazide abolished the effect of (OH)Trp but not that of dihydroergotamine. In conclusion: (i) locally generated 5-HT blunts the inhibitory effect of PTH on Na–Pi co-transport in OK cells; (ii) endogenous 5-HT decreases Pi excretion in rats; and (iii) 5-HT is a paracrine modulator involved in the physiological regulation of renal Pi transport.
Modulation of neutral protease expression in human mesangial cells by hyperglycaemic culture
Authors: ABDEL WAHAB Nadia , MASON Roger M.
Published: August 2015

We report on the effect of prolonged hyperglycaemic (11 and 30 mM d-glucose) culture conditions on human mesangial cell matrix metalloproteinases (MMPs), plasminogen activators and their inhibitors. The results indicate that hyperglycaemic conditions modulate the potential proteolytic activity of the enzymes secreted by confluent cultures of these cells. Gelatinase A (MMP-2) activity was always higher in cultures maintained under hyperglycaemic than under normoglycaemic conditions (4 mM d-glucose). In contrast, gelatinase B (MMP-9) activity was decreased under the same conditions. Matrilysin (MMP-7) activity was decreased by up to 100% under hyperglycaemic conditions. Reverse transcriptase–PCR and Western-blotting analyses indicate that in all cases both the transcripts and the protein level were correlated with enzymic activity.One tissue inhibitor of metalloproteinases, TIMP-2, was barely detectable under hyperglycaemic conditions (30 mM d-glucose). In contrast, TIMP-1 increased during the initial 2 weeks of culture in hyperglycaemic conditions and remained elevated to the end of the experiment (4 weeks). Under normoglycaemic conditions TIMP-1 decreased after 2 weeks of culture. Hyperglycaemic conditions also decreased markedly the activity of tissue plasminogen activator (t-PA). This seemed to be due to increased synthesis of its inhibitor, plasminogen activator inhibitor 1, under these conditions rather than to decreased expression of the t-PA enzyme.
Adenosylcobalamin-dependent glutamate mutase: properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
Authors: HOLLOWAY Daniel E. , HARDING Stephen E. , MARSH E. Neil G.
Published: August 2015

Adenosylcobalamin-dependent glutamate mutase (EC 5.4.99.1) from Clostridium tetanomorphum comprises two protein components, MutE and MutS. The formation of the holoenzyme is a kinetically complex process that involves the co-operative association of MutS, MutE and adenosylcobalamin. The MutS portion of the cobalamin-binding site is conserved within a group of adenosylcobalamin-dependent enzymes that catalyse similar isomerizations. However, in contrast with glutamate mutase, in these other enzymes the cobalamin-binding region represented by MutS is present as a C-terminal domain. We have investigated the effect on the structural and kinetic properties of glutamate mutase of linking MutS to the C-terminus of MutE. Kinetic analysis of this protein, MutES, showed, unexpectedly, that enzyme activity was still co-operatively dependent on protein concentration. The Km for l-glutamate was unchanged from the wild type, whereas Vmax was decreased to approx. one-thirtieth and the Km for coenzyme increased approx. 10-fold. Investigation of the quaternary structure of MutES by equilibrium ultracentrifugation indicated that the protein existed in equilibrium between monomeric and dimeric forms. Thus linking MutE and MutS together seems to substantially weaken the contacts that are responsible for the dimerization of MutE. The two domains of the MutES monomer seem unable to communicate, so that active enzyme is formed by the intermolecular association of two MutES subunits in a co-operative manner.
Evidence for regulated dimerization of cell-cell adhesion molecule (C-CAM) in epithelial cells
Authors: HUNTER Irene , SAWA Hiroki , EDLUND Magnus , ÖBRINK Björn
Published: August 2015

C-CAM is a Ca2+-independent cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily. Addition of chemical cross-linkers to isolated rat liver plasma membranes, intact epithelial cells and purified preparations of C-CAM stabilized one major C-CAM-containing product whose apparent molecular mass was approximately twice that of the C-CAM monomer. The failure to detect additional proteins after cleavage of the cross-linked species demonstrated that C-CAM exists as non-covalently linked dimers both in solution and on the cell surface. Dimerization occurred to the same extent in adherent monolayers and in single cell populations, indicating that dimer formation was the result of cis- interactions within the membranes of individual cells. Using isoform-specific anti-peptide antibodies, both C-CAM1 and C-CAM2 were found to be involved in dimerization, forming predominantly homo-dimeric species. Both calmodulin and Ca2+ ionophore modulated the level of dimer formation, suggesting a role for regulated self-association in the functional activity of C-CAM.
Reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress through the glycation reaction
Authors: KANETO Hideaki , FUJII Junichi , MYINT Theingi , MIYAZAWA Nobuko , ISLAM Kazi N. , KAWASAKI Yoshimi , SUZUKI Keiichiro , NAKAMURA Masahiro , TATSUMI Haruyuki , YAMASAKI Yoshimitsu , TANIGUCHI Naoyuki
Published: August 2015

Several reducing sugars brought about apoptosis in isolated rat pancreatic islet cells and in the pancreatic β-cell-derived cell line HIT. This apoptosis was characterized biochemically by internucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatin condensation and apoptotic body formation. N-Acetyl-l-cysteine, an antioxidant, and aminoguanidine, an inhibitor of the glycation reaction, inhibited this apoptosis. We also showed directly that proteins in β-cells were actually glycated by using an antibody which can specifically recognize proteins glycated by fructose, but not by glucose. Furthermore, fluorescence-activated cell sorting analysis using dichlorofluorescein diacetate showed that reducing sugars increased intracellular peroxide levels prior to the induction of apoptosis. Levels of carbonyl, an index of oxidative modification, and of malondialdehyde, a lipid peroxidation product, were also increased. Taken together, these results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress mainly through the glycation reaction, which may explain the deterioration of β-cells under conditions of diabetes.
Novel isoform of syntaxin 1 is expressed in mammalian cells
Authors: JAGADISH Mittur N. , TELLAM Judy T. , MACAULAY S. Lance , GOUGH Keith H. , JAMES David E. , WARD Colin W.
Published: August 2015

Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neurotoxin C cleavage site (Lys253-Ala254), a feature of the syntaxin 1A protein, because of the novel C-terminal domain of 34 residues. The new C-terminal region contained a single cysteine residue and was moderately rich in proline, with three repeats of a PXP motif. The insert occurred within the region encoding the coiled-coil motifs required for interactions with synaptobrevin, α-SNAP (SNAP being soluble N-ethylmaleimide-sensitive factor attachment protein) and n-Sec1/Munc-18 (n-Sec1 being the rat brain homologue of yeast Sec1p and Munc-18 the mammalian homologue of Caenorhabditis elegans unc-18, but five residues outside the domain previously mapped as being required for binding SNAP-25. Interaction studies in vitro suggested that unlike syntaxin 1A, which binds to both Munc-18a and -18b, syntaxin 1C binds only to Munc-18b. The new isoform syntaxin 1C, which might be generated by alternative splicing of the syntaxin 1 gene, was expressed in several human tissues, including brain. Immunoprecipitation and immunoblotting with the monoclonal antibody HPC-1 and a polyclonal antibody raised against a peptide corresponding to the unique C-terminal 35 residues of syntaxin 1C failed to detect syntaxin 1C at the protein level in extracts of muscle, fat or brain.
Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant
Authors: ERDOGAN Suat , HOUSLAY Miles D.
Published: August 2015

The cAMP phosphodiesterase (PDE) 3 and PDE4 isoforms provide the major cAMP-hydrolysing PDE activities in Jurkat T-cells, with additional contributions from the PDE1 and PDE2 isoforms. Challenge of cells with the adenylate cyclase activator forskolin led to a rapid, albeit transient, increase in PDE3 activity occurring over the first 45 min, followed by a sustained increase in PDE3 activity which began after ∼3 h and continued for at least 24 h. Only this second phase of increase in PDE3 activity was blocked by the transcriptional inhibitor actinomycin D. After ∼3 h of exposure to forskolin, PDE4 activity had increased, via a process that could be inhibited by actinomycin D, and it remained elevated for at least a 24 h period. Such actions of forskolin were mimicked by cholera toxin and 8-bromo-cAMP. Forskolin increased intracellular cAMP concentrations in a time-dependent fashion and its action was enhanced when PDE induction was blocked with actinomycin D. Reverse transcription (RT)-PCR analysis, using generic primers designed to detect transcripts representing enzymically active products of the four PDE4 genes, identified transcripts for PDE4A and PDE4D but not for PDE4B or PDE4C in untreated Jurkat T-cells. Forskolin treatment did not induce transcripts for either PDE4B or PDE4C; however, it reduced the RT-PCR signal for PDE4A transcripts and markedly enhanced that for PDE4D transcripts. Using RT-PCR primers for PDE4 splice variants, a weak signal for PDE4D1 was evident in control cells whereas, in forskolin-treated cells, clear signals for both PDE4D1 and PDE4D2 were detected. RT-PCR analysis of the PDE4A species indicated that it was not the PDE4A isoform PDE-46 (PDE4A4B). Immunoblotting of control cells for PDE4 forms identified a single PDE4A species of ∼118 kDa, which migrated distinctly from the PDE4A4B isoform PDE-46, with immunoprecipitation analyses showing that it provided all of the PDE4 activity in control cells. Forskolin treatment led to a marked decrease of this novel PDE4A species and allowed the detection of a strong signal for an ∼67 kDa PDE4D species, suggested to be PDE4D1, but did not induce PDE4B and PDE4C isoforms. Elevation of intracellular cAMP concentrations in Jurkat T-cells thus exerts a highly selective effect on the transcriptional activity of the genes encoding the various PDE4 isoforms. This leads to the down-regulation of a novel PDE4A splice variant and the induction of PDE4D1 and PDE4D2 splice variants, leading to a net increase in the total PDE4 activity of Jurkat T-cells.
Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes
Authors: NICULESCU Liliana , VEIGA-da-CUNHA Maria , SCHAFTINGEN Emile Van
Published: August 2015

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, d-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human β-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase–regulatory-protein complex either by binding to the regulatory protein (fructose 1-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells.