bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox
Authors: WIENTJES Frans B. , PANAYOTOU George , REEVES Emer , SEGAL Anthony W.
Published: August 2015

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47phox and p67phox, and one in p40phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67phox and p40phox. We have investigated the interaction between p40phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40phox and p67phox where the binding was between the N-terminal half of p67phox and the C-terminal half of p40phox, and did not appear to involve SH3 domains and proline-rich sequences. p40phox also bound p47phox but more weakly than it did p67phox.
Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes
Authors: SUJU Meylin , DAVILA Marbelly , POLEO German , DOCAMPO Roberto , BENAIM Gustavo
Published: August 2015

Phosphatidylethanol is formed by ‘transphosphatidylation’ of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca2+-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca2+-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidylalcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca2+-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca2+ concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca2+-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.
Effects of insulin on the regulation of branched-chain α-keto acid dehydrogenase E1α subunit gene expression
Authors: COSTEAS Paul A , CHINSKY Jeffrey M.
Published: August 2015

Alterations in dietary intake, especially of protein, may produce changes in the hepatic levels of the branched-chain α-keto acid dehydrogenase (BCKAD) complex. The possible role of insulin in the regulation of these observed changes in hepatic capacity for BCKAD expression was therefore examined. Steady-state RNA levels encoding three of the subunits, E1α, E1β and E2, increased by 2–4-fold in the livers of mice starved for 3 days, a known hypoinsulinaemic state. In contrast, the levels of E1β and E2, but not E1α, RNA were decreased when mice were fed 0% protein diets compared with the levels observed in mice fed standard (23%) or higher protein isocaloric diets. BCKAD subunit protein levels under these conditions changed co-ordinately even though the changes in RNA were not co-ordinate. The effects of hormonal changes that might be associated with these dietary changes were examined, using the rodent hepatoma cell line H4IIEC3. In these cells, the levels of E1α protein and mRNA were significantly depressed in the presence of insulin. In contrast, the levels of E1β and E2 RNAs were not decreased by insulin. The half-lives of the E1α and E2 RNAs were determined to be quite long, from 13 to 18 h, with insulin having no dramatic overall effect on the half-lives determined over 24 h. Therefore, it is likely that insulin directly affects the transcription of the E1α gene rather than RNA stability in exerting its negative regulatory effect. This effect is specific to the E1α subunit. The differences in BCKAD subunit RNA levels observed under various nutritional and developmental conditions may therefore be the result of the differential effects of insulin and other hormones on the multiple regulatory mechanisms modulating BCKAD subunit expression.
The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates
Authors: LIBONATI Massimo , BERTOLDI Mariarita , SORRENTINO Salvatore
Published: August 2015

Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40–50% acetic acid solutions. The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides. Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates. In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer. These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.
Down-regulation of cytokine-induced cyclo-oxygenase-2 transcript isoforms by dexamethasone: evidence for post-transcriptional regulation
Authors: RISTIMÄKI Ari , NARKO Kirsi , HLA Timothy
Published: August 2015

The immediate-early cyclo-oxygenase-2 (Cox-2) gene encodes an inducible prostaglandin synthase enzyme that has been implicated in inflammatory and proliferative diseases. We have shown that the inflammatory cytokine interleukin-1 (IL-1) induces the Cox-2 gene in a sustained manner and that post-transcriptional mRNA stabilization is an important event [Ristimäki, Garfinkel, Wessendorf, Maciag and Hla (1994) J. Biol. Chem. 269, 11769–11775]. The anti-inflammatory glucocorticoid dexamethasone potently down-regulates IL-1-induced Cox-2 mRNA expression. Kinetic studies suggest that antagonism of IL-1-induced mRNA stabilization is, at least in part, responsible for the suppression of Cox-2 mRNA. The Cox-2 gene produces two major transcript isoforms, namely Cox-24.6 (4.6 kb) and Cox-22.8 (2.8 kb), which are derived by alternative polyadenylation in the 3´-untranslated region (UTR). In response to dexamethasone, the short Cox-22.8 transcript isoform, which lacks a highly conserved AU-rich region, decays with a longer half-life than the Cox-24.6 isoform. Furthermore, heterologous expression of the hybrid Cox-1 open reading frame and the Cox-2 3´-UTR results in the accumulation of high levels of the short isoform and lower levels of the long isoform. These data suggest that multiple elements in the 3´-UTR of the Cox-2 gene are involved in the determination of the differential mRNA stabilities of Cox-2 transcript isoforms. Because dexamethasone destabilizes the Cox-2 transcript, and because the decay of Cox-2 transcript isoforms induced by dexamethasone occurs with different half-lives, post-transcriptional mRNA destabilization may be an important mechanism in the action of anti-inflammatory glucocorticoids.
Betamethasone modulation of sphingomyelin hydrolysis up-regulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung
Authors: MALLAMPALLI Rama K , MATHUR Satya N , WARNOCK Lorna J , SALOME Ronald G , HUNNINGHAKE Gary W , FIELD F. Jeffrey
Published: August 2015

Glucocorticoids appear to play an integral role in stimulating surfactant synthesis by activating the rate-regulatory enzyme for phosphatidylcholine synthesis, CTP:cholinephosphate cytidylyltransferase (CT). The activity of liver CT, in vitro, has been shown to be inhibited by the sphingomyelin hydrolysis product, sphingosine. In order to investigate the mechanisms by which glucocorticoids alter CT activity, in vivo, we administered betamethasone (1 mg/kg intraperitoneally) sequentially to adult male rats for 5 days. Betamethasone increased CT activity 2-fold relative to control in whole lung. The hormone also increased membrane-bound activity, but did not affect cytosolic enzyme activity. Betamethasone modestly increased CT mRNA as determined by the reverse-transcription PCR and Southern analysis of PCR products, but did not alter the levels of immunoreactive enzyme in lung membranes as demonstrated by Western blotting. The hormone did, however, produce a nearly 3-fold increase in membrane-associated sphingomyelin, and co-ordinately a substantial decrease in the levels of sphingosine in lung membranes. Sphingosine, but not sphinganine, was a competitive, reversible inhibitor of lung CT with respect to the enzyme activator, phosphatidylglycerol. Betamethasone decreased the activities of the sphingomyelin hydrolases: acid sphingomyelinase by 33% and of alkaline ceramidase by 21%. The hormone also inhibited the generation of sphingosine from lysosphingomyelin in lung membranes. There was no significant effect of the hormone on serine palmitoyltransferase activity, the first committed enzyme for sphingolipid biosynthesis. Further, administration of l-cycloserine, an inhibitor of sphingosine formation, was shown to stimulate CT activity by 74% and increase disaturated phosphatidylcholine in alveolar lavage by 52% relative to control. These observations suggest that glucocorticoids up-regulate surfactant synthesis at the level of a key regulatory enzyme by significantly altering the availability of inhibitory metabolites resulting from sphingomyelin hydrolysis.
Location of essential sequence elements at the Escherichia coli melAB promoter
Authors: KEEN Jennifer , WILLIAMS Jacqueline , BUSBY Stephen
Published: August 2015

The Escherichia coli melAB promoter has been cloned on a short DNA fragment and subjected to deletion mutagenesis, random mutagenesis and site-directed mutagenesis. In previous work we had shown that expression from the melAB promoter is triggered by melibiose and that this requires the MelR transcription activator. Melibiose-dependent expression is suppressed by deletions that remove both DNA-binding sites for MelR and by point mutations in the -10 hexamer, the -35 hexamer and the region just upstream of the -35 hexamer. The point mutations identify promoter elements that are essential for triggering the melAB promoter. The importance of these elements was confirmed by site-directed mutagenesis. The results show that the organization of the melAB promoter is fundamentally different from the organization of other bacterial promoters controlled by homologues of MelR.
Functional molecular mass of rat hepatic lipase in liver, adrenal gland and ovary is different
Authors: SCHOONDERWOERD Kees , HOM Marinus L. , LUTHJENS Lee H. , VIEIRA van BRUGGEN Delfina , JANSEN Hans
Published: August 2015

Lipoprotein lipase (LPL) is functionally active only as a dimer. It is also generally assumed that the highly homologous hepatic lipase functions as a dimer, but no clear evidence has been presented. A hepatic lipase-like activity, also indicated as L-type lipase, is present in adrenal and ovary tissues. This enzyme is thought to originate from the liver and to be identical to hepatic lipase. We determined the functional molecular mass of hepatic lipase in rat liver, adrenal gland and ovary by radiation inactivation, a method for determining the functional size of a protein without the need of prior purification. Samples were exposed to ionizing radiation at -135 °C. Hepatic lipase activity in liver homogenate showed a single exponential decay. The functional molecular mass was calculated to be 63±10 kDa. Hepatic lipase activity in adrenal homogenate was found to have a functional molecular mass of 117±16 kDa. The functional molecular masses of the lipases partially purified from rat liver perfusate, adrenal homogenate or ovarian homogenate showed the same pattern, a target mass for the liver enzyme of 56±6 kDa and a target mass of 117±14 kDa for the enzyme from adrenal gland or ovary. In Western blot analysis the mass of the structural units of hepatic lipase in liver was 57 kDa and in adrenal and ovary tissue 51 kDa. We conclude that the functional unit of hepatic lipase in the liver is a monomer. The enzyme in adrenal gland and ovary is different from the liver and the functional unit may be a dimer.
A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells
Authors: GROSCHNER Klaus , SCHUHMANN Klaus , MIESKES Gottfried , BAUMGARTNER Werner , ROMANIN Christoph
Published: August 2015

The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of ‘mode 2’ gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of ‘mode 2’ gating. This effect of PP2Ac was completely prevented by 1 µM okadaic acid. The catalytic subunit of PP1 (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.
Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α2-macroglobulin, rat α1-inhibitor-3 and chemically modified derivatives
Authors: GRØN Hanne , THØGERSEN Ida B. , ENGHILD Jan J. , PIZZO Salvatore V
Published: August 2015

The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human α2-macroglobulin (α2M) and monomeric rat α1-inhibitor-3 (α1 I3). When employing NH3 as the nucleophile, the thiol ester in α1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, α2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for α2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α2M is restored when the proteinase-inhibitory capacity is recovered.
eIF2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2. Sequence conservation between the α, β and δ subunits of eIF2B from mammals and yeast
Authors: PRICE Nigel T. , MELLOR Harry , CRADDOCK Bridget L. , FLOWERS Kevin M. , KIMBALL Scot R. , WILMER Tamsin , JEFFERSON Leonard S. , PROUD Christopher G.
Published: August 2015

The guanine nucleotide-exchange factor eIF2B mediates the exchange of GDP bound to translation initiation factor eIF2 for GTP. This exchange process is a key regulatory step for the control of translation initiation in eukaryotic organisms. To improve our understanding of the structure, function and regulation of eIF2B, we have obtained and sequenced cDNA species encoding all of its five subunits. Here we report the sequences of eIF2Bβ and Δ from rat. This paper focuses on sequence similarities between the α, β and Δ subunits of mammalian eIF2B. Earlier work showed that the amino acid sequences of the corresponding subunits of eIF2B in the yeast Saccharomyces cerevisiae (GCN3, GCD7 and GCD2) exhibit considerable similarity. We demonstrate that this is also true for the mammalian subunits. Moreover, alignment of the eIF2Bα, β and Δ sequences from mammals and yeast, along with the sequence of the putative eIF2Bα subunit from Caenorhabditis elegans and eIF2BΔ from Schizosaccharomyces pombe shows that a large number of residues are identical or conserved between the C-terminal regions of all these sequences. This strong sequence conservation points to the likely functional importance of these residues. The implications of this are discussed in the light of results concerning the functions of the subunits of eIF2B in yeast and mammals. Our results also indicate that the large apparent differences in mobility on SDS/PAGE between eIF2Bβ and Δ subunits from rat and rabbit are not due to differences in their lengths but reflect differences in amino acid composition. We have also examined the relative expression of mRNA species encoding the α, β, Δ and ϵ subunits of eIF2B in a range of rat tissues by Northern blot analysis. As might be expected for mRNA species encoding subunits of a heterotrimeric protein, the ratios of expression levels of these subunits to one another did not vary between the different rat tissues examined (with the possible exception of liver). This represents the first analysis of the levels of expression of mRNA species encoding the different subunits of eIF2B.
Interleukin 1β and cAMP trigger the expression of GTP cyclohydrolase I in rat renal mesangial cells
Authors: PLÜSS Christoph , WERNER Ernst R. , BLAU Nenad , WACHTER Helmut , PFEILSCHIFTER Josef
Published: August 2015

Endogenous synthesis of tetrahydrobiopterin (BH4) is an important requirement for cytokine-stimulated nitric oxide (NO) production in mesangial cells. We have shown that inducible NO synthase is expressed in mesangial cells in response to two principal classes of activating signals, inflammatory cytokines such as interleukin 1β (IL-1β) and agents that elevate cellular levels of cAMP [Kunz, Mühl, Walker and Pfeilschifter (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5387–5391]. In the present paper we demonstrate that IL-1β and cAMP similarly increase the steady-state mRNA levels of GTP cyclohydrolase I (EC 3.5.4.16), the rate-limiting enzyme in BH4 biosynthesis, as measured by a sensitive and quantitative nuclease protection assay. Stimulation of cells with a combination of IL-1β plus cAMP revealed an additive induction profile of GTP cyclohydrolase I mRNA. Message stability studies established that GTP cyclohydrolase I mRNA induced by cAMP has a longer half-life than the IL-1β-induced message. Moreover, cAMP exposure markedly prolonged the half-life of GTP cyclohydrolase I mRNA, from 1.5 to 3.4 h. In a next step we generated a rabbit polyclonal antibody against rat GTP cyclohydrolase I expressed in Escherichia coli and demonstrated that IL-1β and cAMP elevated GTP cyclohydrolase I protein levels in mesangial cells. Furthermore, IL-1β and cAMP led to a marked increase in GTP cyclohydrolase I activity and to increased accumulation of biopterin in mesangial cells. Combinations of IL-1β and cAMP resulted in a synergistic stimulation of GTP cyclohydrolase I activity. This may suggest that, in addition to transcriptional and post-transcriptional regulation, there is a prominent post-translational modulation of enzyme activity.
The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin
Authors: BRINDLE Nicholas P. J. , HOLT Mark R. , DAVIES Joanna E , PRICE Caroline J , CRITCHLEY David R.
Published: August 2015

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actin-filament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-1 (residues 839–850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.
Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves
Authors: HERBERT Derek , PRICE Lindsey J , ALBAN Claude , DEHAYE Laure , JOB Dominique , COLE David J. , PALLETT Kenneth E. , HARWOOD John L.
Published: August 2015

The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and Pi products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (napp) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K´ from the Hill equation) for ACCase1 were 0.054 µM for quizalofop and 21.8 µM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the napp values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K´ values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 µM (ACCase2) and 50 µM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25–10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed.
Biochemical isolation of a membrane microdomain from resting platelets highly enriched in the plasma membrane glycoprotein CD36
Authors: DORAHY Douglas J , LINCZ Lisa F , MELDRUM Clifford J , BURNS Gordon F
Published: August 2015

Here we describe the isolation and characterization of a Triton X-100-insoluble fraction isolated from lysates of platelets by flotation in sucrose gradients. Transmission electron microscopy of the insoluble material revealed a heterogeneous population of vesicles ranging in size from 20 to 1000 nm, and Western blot analyses of platelet lysates for the caveolae structural coat protein, caveolin/VIP21, were negative. Biochemical characterization of the Triton X-100-insoluble fraction showed it to be cholesterol-rich, greatly and specifically enriched in the plasma membrane glycoprotein CD36, and also to contain Src and the Src-related kinase, Lyn. CD36 within this fraction is shown to be palmitoylated, but the fraction itself is not generally enriched in palmitoylated platelet proteins. These results suggest that this fraction represents caveolin-negative, CD36-rich microdomains in the resting platelet membrane. CD36 can form associations with certain Src-related kinases and can signal to activate platelets. These results suggest the possibility that such microdomains are implicated in platelet activation.
Cloning and expression of a new HOXC6 transcript encoding a repressing protein
Authors: CHARIOT Alain , CASTRONOVO Vincent , LE Phuonc , GILLET Claudette , SOBEL Mark E. , GIELEN Jacques
Published: August 2015

Homeodomain-containing proteins are transcription factors that regulate the co-ordinated expression of multiple genes involved in development, differentiation and malignant transformation. In an attempt to characterize expressed homeobox (HOX) genes in breast cancer cells, we cloned two distinct HOXC6 transcripts from an MCF7 cDNA library. Interestingly, one of them represents a new HOXC6 mRNA encoding a homeodomain-containing protein harbouring a unique N-terminal sequence. Moreover we demonstrate that this HOXC6 transcript is less abundant in human breast cancer cells than in non-tumorigenic cell lines, is detected in breast carcinomas and adjacent tissues and is expressed in a variety of human tumours. In addition, transient co-transfection experiments illustrated that both HOXC6 transcripts encode gene products that repress transcription from a HOX binding sequence in MDA-MB231 cells and co-operate with other HOX gene products such as HOXB7 on their target genes. Taken together, our results suggest that HOXC6 proteins might contribute to the breast cell phenotype through co-operative interactions with other HOX-derived proteins and repression of their target genes.
Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants
Authors: BERKEL Patrick H. C. van , VEEN Harry A. van , GEERTS Marlieke E. J. , BOER Herman A. de , NUIJENS Jan H.
Published: August 2015

Human lactoferrin (hLF) is a glycoprotein involved in the host defence against infection and excessive inflammation. Our objective was to determine to what extent each of the three sequons for N-linked glycosylation in hLF is actually used. Human kidney-derived 293(S) cell lines expressing recombinant hLF (rhLF) or glycosylation-site mutants were produced. The mutations involved replacement of asparagine residues with glutamine at one or more sequons for N-glycosylation (Asn138, Asn479 and Asn624). Comparative SDS/PAGE analyses of rhLF, mutated rhLF and human-milk-derived (natural) hLF led us to propose that glycosylation of hLF occurs at two sites (at Asn138 and Asn479) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asn479) or at all three sites occurs in approx. 5% and 9% of hLF respectively. The extent of glycosylation at Asn624 was increased to approx. 29% and 40% of Asn479 and Asn138/479 mutant molecules respectively, which indicates that glycosylation at Asn624 in natural hLF might be limited by glycosylation at Asn479. The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asn138 and Asn479 suggests that glycosylation of hLF is not an absolute requirement for its secretion. The pronounced degradation of unglycosylated hLF in supernatant after mutation at all three glycosylation sites (Asn138/479/624 mutant) but not after mutation at both Asn138 and Asn479 suggests that an altered conformation rather than the lack of glycosylation has rendered the Asn138/479/624 mutant susceptible to intra- and/or extra-cellular degradation.
Structural requirements for annexin I-S100C complex-formation
Authors: SEEMANN Joachim , WEBER Klaus , GERKE Volker
Published: August 2015

S100C is a member of the S100 family of EF-hand-type Ca2+-binding proteins which are thought to bind to and thereby regulate the activity of cellular target proteins in a Ca2+-dependent manner. An intracellular ligand for S100C is the Ca2+/phospholipid-binding protein annexin I and we show here that complex-formation is mediated through unique domains within S100C and annexin I. Using a proteolytically truncated annexin I derivative as well as a number of N-terminal annexin I peptides in liposome co-pelleting and ligand-blotting assays we map the S100C-binding site to the N-terminal 13 residues of annexin I. Similar analyses employing recombinantly expressed S100C mutants reveal that residues D91 to I94 in the unique C-terminal extension of this S100 protein are indispensable for annexin I binding. Interaction between S100C and an N-terminal annexin I peptide containing a tryptophan at position 11 can also be monitored by fluorescence emission spectroscopy after tryptophan excitation. This analysis indicates that the local environment of the tryptophan in annexin I becomes less aqueous on S100C binding, suggesting a hydrophobic nature of the protein-protein interaction. Thus the structural basis of the annexin I-S100C complex-formation probably resembles to a large extent that of the well-characterized annexin II-p11 interaction.
Role of lysine, tryptophan and calcium in the β-elimination activity of a low-molecular-mass pectate lyase from Fusarium moniliformae
Authors: RAO M. Narsimha , KEMBHAVI Asha A. , PANT Aditi
Published: August 2015

An extracellular pectate lyase from Fusarium moniliformae was purified to homogeneity by affinity chromatography followed by gel filtration, with a yield of 76.5%. Laser desorption MS of the enzyme gave a molecular mass of 12133.5±2.5 Da. The pectate lyase was a glycoprotein with a 5% carbohydrate content and had a pI value of 9.1. Atomic-emission spectrometry showed that Ca2+ was a part of the holoenzyme held by carboxy groups of the protein. These results support the hypothesis of a putative Ca2+ site suggested by Yodder, Keen and Jurnak [(1993) Science 260, 1503–1507] in the crystal structure of pectate lyase C of Erwinia chrysanthemi. Loss of Ca2+ was observed by treatment with EGTA or carboxy-modifying Woodward's reagent K, with subsequent loss of enzyme activity. Tryptophan fluorescence quenching showed that Ca2+ does not affect binding of substrate to enzyme. Chemical-modification and substrate-protection studies showed the presence of lysine and tryptophan at or near the active site of the pectate lyase. Chemically modified enzyme showed no major structural changes as determined by CD. Amino acid analyses of native, trinitrobenzenesulphonate (TBNS)-treated and substrate-protected TNBS-treated enzyme showed that a single essential residue of lysine is present at or near the active-site. Substrate-affinity studies showed that tryptophan could be essential for substrate binding, whereas lysine could be involved in the catalysis. Fluorescence quenching further confirmed the involvement of tryptophan in substrate binding. The reaction mechanism involving β-elimination by this enzyme is discussed.
Thyromimetic mode of action of peroxisome proliferators: activation of ‘malic’ enzyme gene transcription
Authors: HERTZ Rachel , NIKODEM Vera , BEN-ISHAI Alumit , BERMAN Inna , BAR-TANA Jacob
Published: August 2015

Peroxisome proliferators induce thyroid-hormone-dependent liver activities, e.g. ‘malic’ enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, S14 [Hertz, Aurbach, Hashimoto and Bar-Tana (1991) Biochem. J. 274, 745–751]. Here we report that the thyromimetic effect of peroxisome proliferators with respect to ‘malic’ enzyme results from transcriptional activation of the ‘malic’ enzyme gene, mediated by binding of the peroxisome proliferator activated receptor (PPARα)/retinoid X receptor (RXRα) heterodimer to a 5´-flanking enhancer of the ‘malic’ enzyme promoter. The enhancer involved is distinct from the thyroid hormone response element of the ‘malic’ enzyme promoter and is partly homologous with that which mediates transcriptional activation of peroxisomal acyl-CoA oxidase by peroxisome proliferators. Hence transcriptional activation of thyroid-hormone-dependent liver genes by xenobiotic or endogenous amphipathic carboxylates collectively defined as peroxisome proliferators is mediated by a transduction pathway similar to that involved in transcriptional activation of peroxisomal β-oxidative genes and distinct from that which mediates thyroid hormone action.
Tissue-specific distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA- and ADP-ribosylation factor (ARF)-regulated isoenzymes
Authors: PROVOST Joseph J , FUDGE Jennifer , ISRAELIT Sallie , SIDDIQI Abdur R , EXTON John H.
Published: August 2015

Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5´-[γ-thio]triphosphate (GTP[S]), with the highest specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.
A method for determination in situ of variations within the hepatic lobule of hepatocyte function and metabolite concentrations
Authors: BURNS Shamus P , COHEN Robert D. , ILES Richard A , GERMAIN Jocelyn P. , GOING Thomas C. H. , EVANS Stephen J. W. , ROYSTON Patrick
Published: August 2015

A method is described for the production of detailed maps of intralobular variations of hepatocyte function and metabolite concentrations, based on variable destruction by digitonin of the lobule from the centrilobular direction. Instead of the conventional approach, in which isolated hepatocytes are then prepared and studied in suspension, perfusion is continued after digitonin treatment and the function of the unaffected lobular remnants is determined, or mean metabolite concentrations are measured by 31P-NMR. These measurements are plotted against the degree of destruction, determined precisely after each study by automated quantitative histomorphometry. These plots are transformed into curves of the function or metabolite concentration of nominal single cells at any point along the radius of the lobule. Gluconeogenesis from lactate remained stable, although reduced, even after 85–90% lobular destruction, predominated periportally and disappeared by 50% along the radius of the lobule. In 31P-NMR studies, employing 1.5 mM lactate as substrate, narrowing of the intracellular Pi resonance was observed as digitonin destruction increased; this was attributed to a decrease in the intralobular heterogeneity of the intracellular pH, which fell from approx. 7.9 to < 7.4 along the first 16% of the lobular radius (from the periportal end) and to < 7.3 in the remainder of the lobule. The ATP concentration rose, and then fell, along the radius of the lobule in a centripetal direction. The method is potentially generally applicable to a wide range of hepatocellular functions and to the measurement of metabolite concentrations, most conveniently those susceptible to estimation by NMR.
Lysophosphatidic acid-sensitive intracellular Ca2+ store does not regulate Ca2+ entry at plasma membrane in Jurkat human T-cells
Authors: TAKEMURA Haruo , IMOTO Kenshi , SAKANO Shoji , KANEKO Masamitsu , OHSHIKA Hideyo
Published: August 2015

In Jurkat T cells, the anti-CD3 antibody OKT3 and thapsigargin (TG) elevated the cytoplasmic free Ca2+ concentration ([Ca2+]i), after which it decreased to a sustained, elevated level. In contrast, lysophosphatidic acid (LPA) increased [Ca2+]i only briefly and transiently, after which it declined to the resting level of [Ca2+]i even in the presence of extracellular Ca2+. OKT3 increased Ins(1,4,5)P3 formation but neither LPA nor TG did. In the absence of extracellular Ca2+, the addition of OKT3 did not affect an elevation of [Ca2+]i induced by the subsequent addition of LPA and vice versa. In permeabilized Jurkat cells, the addition of Ins(1,4,5)P3 released Ca2+; this was inhibited by heparin, whereas LPA released Ca2+ even in the presence of heparin. cADP-ribose released Ca2+; this was additive with LPA-induced Ca2+ release and vice versa in permeabilized Jurkat cells. LPA did not stimulate Ca2+ entry and 45Ca2+ uptake but OKT3 and TG did. LPA, OKT3 and TG did not affect the sustained elevation of [Ca2+]i induced by ionomycin. The present results suggest that at least three kinds of intracellular Ca2+ stores, which are Ins(1,4,5)P3-, cADP-ribose- and LPA-sensitive, exist in Jurkat T cells, and that the LPA-sensitive intracellular Ca2+ store does not regulate Ca2+ entry at the plasma membrane.
Identification and function of type-2 and type-3 ryanodine receptors in gut epithelial cells
Authors: VERMA Vandana , CARTER Christine , KEABLE Susan , BENNETT Deborah , THORN Peter
Published: August 2015

Reverse transcription-PCR (RT-PCR) techniques were used to identify the expression of ryanodine receptor (RyR) isoforms in gut epithelial cells. Restriction digest and sequence analysis of the PCR product showed the presence of RyR 2 and RyR 3. [3H]Ry binding studies on a microsome preparation, in a high-salt buffer, showed specific binding with an EC50 of 15 µM. In order to determine a potential functional role for these RyRs, we first characterized the response of the cells to acetylcholine. At all concentrations used acetylcholine induced sinusoidal cytosolic Ca2+ concentration ([Ca2+]i) oscillations. In response to 10-4 M acetylcholine, levels of inositol 1,4,5-trisphosphate (InsP3) showed a peak of six times the basal level, at 30 s after stimulation. Application of caffeine alone failed to elicit a rise in cytosolic Ca2+. However, caffeine (5–50 mM) did rapidly and reversibly inhibit the acetylcholine-induced [Ca2+]i oscillations. The effects of Ry were more complex. Applied alone, Ry had no effect on the [Ca2+]i signal. When applied during agonist-evoked [Ca2+]i oscillations, Ry (10 µM) slowly blocked the response. In the continuous presence of Ry (10 µM) a short application of acetylcholine elicited a [Ca2+]i response that continued as oscillations even when the agonist was removed. The oscillations, in the presence of Ry (10 µM) but absence of agonist, were blocked either by removal of extracellular Ca2+ or by an application of a higher concentration of Ry (100 µM). These effects are consistent with the known use-dependence and dose-dependence for Ry action at the RyR. We conclude that the RyR 2 and RyR 3, identified by RT-PCR, play a central role in [Ca2+]i oscillations in gut epithelial cells.
Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells
Authors: FLUITER Kees , VIETSCH Helene , BIESSEN Eric A. L. , KOSTNER Gert M. , BERKEL Theo J. C. van , SATTLER Wolfgang
Published: August 2015

Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6±2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4±0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 °C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH.
Rat C6 glioma cell growth is related to glucose transport and metabolism
Authors: NAGAMATSU Shinya , NAKAMICHI Yoko , INOUE Nobuo , INOUE Masae , NISHINO Haruo , SAWA Hiroki
Published: August 2015

In order to establish whether growth of glioma cells is associated with glucose transport and metabolism, we investigated expression of the glucose transporter and hexokinase, as well as glucose transport and glucose phosphorylation in rat C6 glioma cells growing at different rates. Rat C6 glioma cells were subcloned to produce four different cell lines (CL1, CL2, CL3 and CL4) differing in growth, differentiation and morphology: CL1 cells were slow-growing with an astrocytic appearance whereas CL4 cells grew rapidly and were small and spindle-shaped. Immunocytochemical analysis using glial fibrillary acidic protein and galactocerebroside antibodies revealed that CL1 and CL4 cells differentiate to astrocytes and oligodendrocytes respectively. Both of these cell lines expressed GLUT1 mRNA predominantly, whereas little GLUT3 mRNA was evident by Northern-blot analysis. The GLUT1 mRNA level was much higher in CL4 than in CL1 cells, and the uptake of 2-deoxy-D-glucose and 3-O-methyl-D-glucose by CL4 cells was markedly higher than that by CL1 cells, indicating a correlation between the growth rate, glucose transporter (GLUT1) level and glucose-transport rate of C6 glioma cells. We then studied glucose metabolism by CL1 and CL4 cells by measuring their hexokinase activities and intracellular concentrations of glucose and ATP. The mitochondrial hexokinase activity of CL4 cells was about three times higher than that of CL1 cells, whereas the cytosolic hexokinase activity of CL4 cells was only about half that of CL1 cells. As the total amount of cellular hexokinase protein in CL4 cells was only slightly higher (about 20%) than that in CL1 cells, the hexokinase protein of CL4 cells was considered to have moved from the cytosol to the mitochondrial membranes. Consistent with the increased mitochondrial hexokinase activity of CL4 cells, the intracellular glucose concentration was undetectable, and the ATP concentration was higher than that of CL1 cells, suggesting that glucose transport is the rate-limiting factor for overall glucose metabolism in rapidly growing C6 cells. Therefore the present data demonstrate that glioma cell growth is related to glucose transport, which is closely associated with glucose metabolism.
Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases
Authors: CHAVAGNAT Frederic , DUEZ Colette , GUINAND Micheline , POTIN Philippe , BARBEYRON Tristan , HENRISSAT Bernard , WALLACH Jean , GHUYSEN Jean-Marie
Published: August 2015

A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni2+-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.