bluuebunny/crossref_metadata_2025 huggingface dataset:
cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I
Authors: GUERRIN Marina , ISHIGAMI Akihito , MÉCHIN Marie-Claire , NACHAT Rachida , VALMARY Séverine , SEBBAG Mireille , SIMON Michel , SENSHU Tatsuo , SERRE Guy
Published: February 2003

Peptidylarginine deiminases (PADs) catalyse a post-translational modification of proteins through the conversion of arginine residues into citrullines. The existence of four isoforms of PAD (types I, II, III and IV) encoded by four different genes, which are distinct in their substrate specificities and tissue-specific expression, was reported in rodents. In the present study, starting from epidermis polyadenylated RNA, we cloned by reverse transcriptase-PCR a full-length cDNA encoding human PAD type I. The cDNA was 2711bp in length and encoded a 663-amino-acid sequence. The predicted protein shares 75% identity with the rat PAD type I sequence, but displays only 50—57% identity with the three other known human isoforms. We have described the organization of the human PAD type I gene on chromosome 1p36. A recombinant PAD type I was produced in Escherichia coli and shown to be enzymically active. Human PAD type I mRNAs were detected by reverse transcriptase-PCR not only in the epidermis, but also in various organs, including prostate, testis, placenta, spleen and thymus. In human epidermis extracts analysed by Western blotting, PAD type I was detected as a 70kDa polypeptide, in agreement with its predicted molecular mass. As shown by immunohistochemistry, the enzyme was expressed in all the living layers of human epidermis, with the labelling being increased in the granular layer. This is the first description of the human PAD type I gene and the first demonstration of its expression in epidermis.
Factor VII mutant V154G models a zymogen-like form of Factor VIIa
Authors: TOSO Raffaella , BERNARDI Francesco , TIDD Theresa , PINOTTI Mirko , CAMIRE Rodney M. , MARCHETTI Giovanna , HIGH Katherine A. , POLLAK Eleanor S.
Published: February 2003

Proteolytic cleavage of the peptide bond between Arg152 and Ile153 converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile153 and Asp343 drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val154→Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile153 to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly154 is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII—TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in l-α-phosphatidylcholine for the zymogen FVII (Arg152→Gln, Kd = 1.04±0.27nM), inactive FVIIa (Ser344→Ala, Kd = 0.27±0.06nM) and a zymogen-like FVIIa (V154G, Kd = 1.15±0.16nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 105-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.
Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa
Authors: RAO Veena S. , JOSEPH Jeremiah S. , KINI R. Manjunatha
Published: February 2003

Procoagulant venoms of several Australian elapids contain proteinases that specifically activate prothrombin; among these, Group D activators are functionally similar to coagulation factor Xa (FXa). Structural information on this class of prothrombin activators will contribute significantly towards understanding the mechanism of FXa-mediated prothrombin activation. Here we present the purification of Group D prothrombin activators from three Australian snake venoms (Hoplocephalus stephensi, Notechis scutatus scutatus and Notechis ater niger) using a single-step method, and their N-terminal sequences. The N-terminal sequence of the heavy chain of hopsarin D (H. stephensi) revealed that a fully conserved Cys-7 was substituted with a Ser residue. We therefore determined the complete amino acid sequence of hopsarin D. Hopsarin D shows 70% similarity with FXa and 98% similarity with trocarin D, a Group D prothrombin activator from Tropidechis carinatus. It possesses the characteristic Gla domain, two epidermal growth factor-like domains and a serine proteinase domain. All residues important for catalysis are conserved, as are most regions involved in interactions with factor Va and prothrombin. However, there are some structural differences. Unlike FXa, hopsarin D is glycosylated in both its chains: in light-chain residue 52 and heavy-chain residue 45. The glycosylation on the heavy chain is a large carbohydrate moiety adjacent to the active-site pocket. Overall, hopsarin D is structurally and functionally similar to mammalian coagulation FXa.
Conformational change in elastase following complexation with alpha1-proteinase inhibitor: a CD investigation
Authors: BOUSQUET Jean-Alain , DURANTON Jérôme , MÉLY Yves , BIETH Joseph G.
Published: February 2003

The CD spectrum of porcine pancreatic elastase in complex with α1-proteinase inhibitor (α1-PI) was calculated by subtracting the CD spectrum of the proteolytically cleaved inhibitor from that of the elastase—α1-PI complex. Elastase undergoes a moderate secondary structure change: its β-structure is partially disordered while its α-helix content is poorly affected. In contrast, its tertiary structure undergoes a significant structural loosening upon complexation. These alterations have been compared with those following chemical and thermal unfolding of free elastase. Inhibitor-bound elastase and the denaturation intermediate of free elastase share secondary but not tertiary structural features. On the other hand, both free and complexed elastases undergo a single-step transition in tertiary structure upon thermal unfolding. These data are discussed in terms of the inhibition and structural modification of elastase induced by α1-PI observed by previous investigators.
A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition
Authors: CREPIN Valerie F. , FAULDS Craig B. , CONNERTON Ian F.
Published: February 2003

Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties. We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris. The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no. AJ293029). The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (Km 0.048mM, Ki 2.5mM and Vmax 8.2units/mg against methyl 3,4-dihydroxycinnamate). The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action.
Neighbouring bases in template influence base-pairing of isoguanine
Authors: MACIEJEWSKA Agnieszka M. , LICHOTA Katarzyna D. , KUŚMIEREK Jarosław T.
Published: February 2003

Assuming that the efficiency of the incorporation of 5-methyl-2′-deoxyisocytosine-5′ triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3′-TXT-5′, 3′-GXG-5′, 3′-CXC-5′, 3′-CXT-5′, where X = iG) at 37, 50, 65 and 80°C, we found that 3′-neighbours decrease the fraction of the keto tautomer in the order C>GT, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5—20) primarily reflects the variability of Km values, since Vmax values are roughly similar, which indicates that the iG·MiC and iG·T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O2-diphenylcarbamoyl-N6-[(dimethylamino)ethylidene]-2′-deoxyisoguanosine, is extremely labile (t1/2 = 3.5min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.
Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration
Authors: ZANNETTINO Andrew C.W. , ROUBELAKIS Maria , WELLDON Katie J. , JACKSON Denise E. , SIMMONS Paul J. , BENDALL Linda J. , HENNIKER Anthony , HARRISON Kate L. , NIUTTA Silvana , BRADSTOCK Kenneth F. , WATT Suzanne M.
Published: February 2003

SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY246AQL and VVY263ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34+CD38+ and early clonogenic progenitors, and at lower levels on CD34+CD38- cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133+ precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Δ46—110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility.
Interdomain communication in the molecular chaperone DnaK
Authors: HAN Wanjiang , CHRISTEN Philipp
Published: February 2003

DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain. Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP·DnaK and GrpE-liganded ADP·DnaK. Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions. Binding of peptide increased the intrinsic fluorescence of both ATP·DnaK and ADP·DnaK·GrpE and rendered their spectra similar to the spectrum of ADP·DnaK with closed conformation of the ATPase domain. These results, together with the differential kinetics of peptide binding to ADP·DnaK on the one hand, and to ATP·DnaK or ADP·DnaK·GrpE on the other, suggest that ligands for either domain, i.e. ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.
Mechanism of action of interleukin-2 (IL-2)-Bax, an apoptosis-inducing chimaeric protein targeted against cells expressing the IL-2 receptor
Authors: AQEILAN Rami , KEDAR Rotem , BEN-YEHUDAH Ahmi , LORBERBOUM-GALSKI Haya
Published: February 2003

The chimaeric protein interleukin-2 (IL-2)—Bax was designed to target and kill specific cell populations expressing the IL-2 receptor. However, it is not well understood how IL-2—Bax causes target cells to die. In the present study, we investigated the pathway of apoptosis evoked by IL-2—Bax and the possible involvement of endogenous Bax in this process. We report here that, upon internalization of IL-2—Bax into target cells, it is localized first mainly in the nucleus, and only later is it translocated to the mitochondria. Similarly, endogenous Bax is also partially localized in the nucleus, and accumulates mainly in this compartment soon after physiological triggering of apoptosis. Despite the fact that Bax has no nuclear localization sequence, our data suggest that Bax has one or more physiological roles and/or substrates within the nucleus. Indeed, a dramatic repression of nuclear Tax protein expression was induced following treatment of HUT-102 cells with IL-2—Bax, similar to what occurs following serum deprivation of these cells. Unexpectedly, induction of apoptosis using IL-2—Bax was preceded by enhanced expression of newly synthesized Bax protein and suppression of Bcl-2. This imbalance between the pro- and anti-apoptotic genes was associated with p53 induction, although IL-2—Bax activity was also evident in cells lacking p53 expression. By studying the mechanism of action of IL-2—Bax, we were able to follow the intrinsic events and their cascade that culminates in cell death. We have shown that the ability of IL-2—Bax to affect the intracellular apoptotic machinery within the target cells, and to cause the cells to die, uses a mechanism similar to that induced following a normal apoptotic signal.
Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein
Authors: THEKKUMKARA Thomas J. , LINAS Stuart L.
Published: February 2003

The mRNA 3′-untranslated region (3′-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT1A) with and without 3′-UTR differed in AT1A mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT1A 3′-UTR probe identified a major mRNA-binding protein complex of 55kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT1A receptor 3′-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT1A 3′-UTR sequence ‘decoy’ to compete with the native receptor 3′-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT1A receptor and [125I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT1A receptors in WB (Bmax = 2.7pmol/mg of protein, Kd = 0.56nM). Northern-blot analysis showed a significant increase in native receptor mRNA expression in 3′-UTR decoy-expressing cells, confirming the role of 3′-UTR in mRNA destabilization. Compared with vehicle control, AngII induced DNA and protein synthesis in wild-type WB as measured by [3H]thymidine and [3H]leucine incorporation respectively. Activation of [3H]thymidine and [3H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT1 receptor coupling with G-protein αi. We also delineated that functional coupling of AT1A receptor with G-protein αi is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein αi activation. In contrast with wild-type cells, stable expression of the 3′-UTR ‘decoy’ produced AngII-stimulated protein synthesis and cellular hypertrophy as demonstrated by a significant increase in [3H]leucine incorporation and no increase in [3H]thymidine incorporation and cell number. Furthermore, [125I]AngII cross-linking and immunoprecipitation studies using specific G-protein α antibodies showed that in wild-type cells, the AT1A receptor coupled with G-protein αi, whereas in cells expressing the 3′-UTR ‘decoy’, the AT1A receptor coupled with G-protein αq. These findings indicate that the 3′-UTR-mediated changes in receptor function may be mediated in part by a switch from G-protein αi to G-protein αq coupling of the receptor. Our results suggest that the 3′-UTR-mediated post-transcriptional modification of the AT1A receptor is critical for regulating tissue-specific receptor functions.
Comparison of nitration and oxidation of tyrosine in advanced human carotid plaque proteins
Authors: MORTON Lincoln W. , PUDDEY Ian B. , CROFT Kevin D.
Published: February 2003

The importance of reactive nitrogen species in atherosclerosis remains poorly understood, despite the semi-quantitative evidence for the presence of 3-nitrotyrosine provided by immunohistochemical staining studies. At this time, there appear to be no data describing the prevalence of nitration relative to oxidation in atherosclerotic plaque proteins. The present study used 3-nitrotyrosine and dityrosine as markers of nitration and oxidation respectively to examine the relative abundance of each process. Substantial methodological improvements were required to overcome problems associated with sensitivity and artefactual production of 3-nitrotyrosine when quantified by GLC-MS. It was shown that careful selection of hydrolysis vessel, sample reduction and the use of the oxazolinone derivative provided sample stability and exquisite sensitivity. Using these methods, it was observed that the frequency of nitration was 92±15μmol/mol of tyrosine (0.01%). Dityrosine was present at 1.5±0.14mmol/mol of tyrosine (0.30%) using HPLC/fluorescence; thus nitration accounted for approx. 3% of the tyrosine modifications measured. Given that other modifications of tyrosine are known to occur in carotid plaque proteins, the contribution of nitration to the total pool of modified tyrosine is very small. However, the possibility of metabolic processes or chemical agents modifying 3-nitrotyrosine to secondary oxidation products remains an alternative explanation for the low levels demonstrated in this study.
Age-related changes in the composition, the molecular stoichiometry and the stability of proteoglycan aggregates extracted from human articular cartilage
Authors: WELLS Terri , DAVIDSON Catherine , MÖRGELIN Matthias , BIRD Joseph L.E. , BAYLISS Michael T. , DUDHIA Jayesh
Published: February 2003

The heterogeneity of the components of proteoglycan aggregates, their stoichiometry within the aggregate and the aggregates’ stability was investigated in normal human articular cartilage specimens (age-range newborn to 63 years). Proteoglycans were extracted from tissue by sequentially extracting them with PBS alone, PBS containing oligosaccharides of hyaluronan, and PBS containing solutions of increasing guanidinium chloride concentration (1M, 2M, 3M and 4M). A high proportion of each of the components of the proteoglycan aggregate, i.e. uronic acid, sulphated glycosaminoglycan, hyaluronan binding domain of aggrecan (G1-domain), link protein (LP) and hyaluronan, was extracted from immature cartilage by PBS alone and PBS containing oligosaccharides of hyaluronan. This was in marked contrast to adult cartilage, which required high concentrations of guanidinium chloride for the efficient extraction of these components. The molar ratios of total G1-domain:LP and the G1-domain associated with aggrecan:LP also differed markedly between immature and mature cartilage and between each of the sequential extracts. The concentration of LP was less than that of the G1-domain in all extracts of cartilage from individuals over 13 years, but this was particularly noticeable in the 1M guanidinium chloride extracts, and it was surmised that a deficiency in LP produces unstable aggregates in situ. The fragmentation of LP, which is known to occur with advancing age, did not influence the extractability of LP, and fragments were present in each of the sequential extracts. Therefore the generally accepted model of proteoglycan aggregation presented in the literature, which is mostly derived from analysis of immature animal cartilage, cannot be used to describe the structure and organization of aggregates in adult human articular cartilage, where a heterogeneous population of complexes exist that have varying degrees of stability.
Ceramide 1-phosphate increases intracellular free calcium concentrations in thyroid FRTL-5 cells: evidence for an effect mediated by inositol 1,4,5-trisphosphate and intracellular sphingosine 1-phosphate
Authors: HÖGBACK Susanna , LEPPIMÄKI Petra , RUDNÄS Britt , BJÖRKLUND Sonja , SLOTTE J. Peter , TÖRNQUIST Kid
Published: February 2003

Sphingolipid (SP) derivatives have diverse effects on the regulation of intracellular free calcium concentrations ([Ca2+]i) in a multitude of non-excitable cells. In the present investigation, the effect of C2-ceramide 1-phosphate (C1P) on [Ca2+]i was investigated in thyroid FRTL-5 cells. C1P evoked a concentration-dependent increase in [Ca2+]i, both in a calcium-containing and a calcium-free buffer. A substantial part of the C1P-evoked increase in [Ca2+]i was due to calcium entry. The effect of C1P was attenuated by overnight pretreatment of the cells with pertussis toxin. Similar results were obtained with C8-ceramide 1-phosphate, although the magnitude of the responses was smaller than with C1P. The phospholipase C inhibitor U73122 attenuated the effect of C1P. C1P invoked a small, but significant, increase in inositol 1,4,5-trisphosphate (IP3). However, the effect of C1P on [Ca2+]i was inhibited by neither Xestospongin C, 2-aminoethoxydiphenylborate nor neomycin. C1P mobilized calcium from an IP3-sensitive calcium store, as C1P did not increase [Ca2+]i in cells pretreated with thapsigargin. The effect of C1P on [Ca2+]i was potently attenuated by dihydrosphingosine and dimethylsphingosine, two inhibitors of sphingosine kinase, but not by the inactive SP-derivative N-acetyl sphingosine. Stimulating the cells with C1P evoked an increase in the production of intracellular sphingosine 1-phosphate. C1P did not modulate DNA synthesis or the forskolin-evoked production of cAMP. The results indicate that C1P may be an important SP participating in cellular signalling.
Peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated regulation of multidrug resistance 2 (Mdr2) expression and function in mice
Authors: KOK Tineke , BLOKS Vincent W. , WOLTERS Henk , HAVINGA Rick , JANSEN Peter L.M. , STAELS Bart , KUIPERS Folkert
Published: February 2003

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that controls expression of genes involved in lipid metabolism and is activated by fatty acids and hypolipidaemic fibrates. Fibrates induce the hepatic expression of murine multidrug resistance 2 (Mdr2), encoding the canalicular phospholipid translocator. The physiological role of PPARα in regulation of Mdr2 and other genes involved in bile formation is unknown. We found no differences in hepatic expression of the ATP binding cassette transporter genes Mdr2, Bsep (bile salt export pump), Mdr1a/1b, Abca1 and Abcg5/Abcg8 (implicated in cholesterol transport), the bile salt-uptake systems Ntcp (Na+-taurocholate co-transporting polypeptide gene) and Oatp1 (organic anion-transporting polypeptide 1 gene) or in bile formation between wild-type and Pparα(-/-) mice. Upon treatment of wild-type mice with ciprofibrate (0.05%, w/w, in diet for 2 weeks), the expression of Mdr2 (+3-fold), Mdr1a (+6-fold) and Mdr1b (+11-fold) mRNAs was clearly induced, while that of Oatp1 (-5-fold) was reduced. Mdr2 protein levels were increased, whereas Bsep, Ntcp and Oatp1 were drastically decreased. Exposure of cultured wild-type mouse hepatocytes to PPARα agonists specifically induced Mdr2 mRNA levels and did not affect expression of Mdr1a/1b. Altered transporter expression in fibrate-treated wild-type mice was associated with a 400% increase in bile flow: secretion of phospholipids and cholesterol was increased only during high-bile-salt infusions. No fibrate effects were observed in Pparα(-/-) mice. In conclusion, our results show that basal bile formation is not affected by PPARα deficiency in mice. The induction of Mdr2 mRNA and Mdr2 protein levels by fibrates is mediated by PPARα, while the induction of Mdr1a/1b in vivo probably reflects a secondary phenomenon related to chronic PPARα activation.
Substrate-induced conformational transition in human phenylalanine hydroxylase as studied by surface plasmon resonance analyses: the effect of terminal deletions, substrate analogues and phosphorylation
Authors: STOKKA Anne J. , FLATMARK Torgeir
Published: February 2003

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of l-phenylalanine (l-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3min at 25°C) with an end point of approx. 75RU (resonance units)/(pmolsubunit/mm2). By contrast, the contribution of binding the substrate (165Da) to its catalytic core enzyme [ΔN(1—102)/ΔC(428—452)-hPAH] was only approx. 2RU/(pmolsubunit/mm2). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S]0.5-value of 98±7μM (h = 1.0) and 158±11μM, respectively, i.e. for the tetramer it is slightly lower than the value (145±5μM) obtained for the co-operative binding (h = 1.6±0.4) of l-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon l-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1—117) plus catalytic domain (residues 118—411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-l-alanine revealed an SPR response comparable with that of l-Phe on binding to wild-type hPAH.
Selenite sensitizes mitochondrial permeability transition pore opening in vitro and in vivo: a possible mechanism for chemo-protection
Authors: SHILO Shani , ARONIS Anna , KOMARNITSKY Rita , TIROSH Oren
Published: February 2003

There is a known connection between selenium supplementation and chemo-protective anti-cancer activity. This biological phenomenon may be due to the ability of selenium to instigate cellular apoptosis. However, the mechanism by which selenium promotes cellular apoptosis is still obscure. The present study shows that sodium selenite, a common dietary form of selenium, promotes the mitochondrial permeability transition (MPT) in isolated rat liver mitochondria both in vitro and following in vivo supplementation. A low selenium concentration (0.1—10μM) strongly induced cyclosporin A-sensitive mitochondrial swelling. Selenium also promoted both calcium release from the matrix of isolated mitochondria and uncoupled respiration. The MPT-inducing effect of selenium provoked the release of cytochrome c, a pro-apoptotic factor, into the incubation medium. Selenium did not increase intra-mitochondrial peroxide production, but did consume endogenous mitochondrial glutathione. Moreover, the effect of MPT induction was greatly potentiated in the presence of thiol-bearing antioxidants, e.g. N-acetylcysteine and lipoamide. During MPT progression, selenium induced NADH oxidation via electron acceptance from complex I. Supplementation for 20 days with 16p.p.m. selenium in the drinking water of rats increased the propensity of mitochondria to undergo the MPT. More marked mitochondrial swelling in response to calcium and lower calcium-uptake capacity were observed, in the absence of liver damage or the intensive oxidation of reduced glutathione. Therefore selenite facilitates MPT pore opening via its thiol- and NADH/complex I-dependent reduction, and thereby may provide chemo-protection by potentiation of the capacity of the mitochondria to regulate programmed cell death. Data from the present study suggest that selenium can regulate important mitochondrial functions both in vivo and in vitro.
Spatial characterisation of ryanodine-induced calcium release in mouse pancreatic acinar cells
Authors: ASHBY Michael C. , PETERSEN Ole H. , TEPIKIN Alexei V.
Published: February 2003

In pancreatic acinar cells, agonists evoke intracellular Ca2+ transients which are initiated in the apical region of these polarized cells. There are contradictory experimental data concerning Ca2+ release from ryanodine receptors (RyRs) in the apical region. In the present study, we have used low doses of ryanodine to open RyRs leading to the release of Ca2+ from intracellular stores. Ryanodine causes Ca2+ release that is initiated in the apical region of the cell but is dependent upon functional inositol 1,4,5-trisphosphate receptors (IP3Rs). These results suggests that co-ordinated release from co-localized RyRs and IP3Rs underlies the increased sensitivity of the apical region to initiation of intracellular Ca2+ transients.
Acetylacetone-cleaving enzyme Dke1: a novel C-C-bond-cleaving enzyme from Acinetobacter johnsonii
Authors: STRAGANZ Grit D. , GLIEDER Anton , BRECKER Lothar , RIBBONS Douglas W. , STEINER Walter
Published: February 2003

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe2+ is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a Km of 9.1μM and a kcat of 8.5s-1. A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.
Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine
Authors: MALYKH Yanina N. , KING Timothy P. , LOGAN Elizabeth , KELLY Denise , SCHAUER Roland , SHAW Lee
Published: February 2003

N-Glycolylneuraminic acid (Neu5Gc), an abundant sialic acid in animal glycoconjugates, is formed by the enzyme CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. The amount of Neu5Gc relative to other sialic acids is highly dependent on the species, tissue and developmental stage. Although the activity of the hydroxylase is a key factor in controlling Neu5Gc incorporation in adult animals, little is known about the regulation of hydroxylase expression and the role of this enzyme in determining changes in Neu5Gc during development. Using pig small intestine as a model system, the appearance of total sialic acid and the regulation of Neu5Gc biosynthesis during development were studied in various regions of this tissue. The amount of total sialic acid and Neu5Gc declined markedly in 2 weeks after birth. Although in subsequent developmental phases there were no positional differences in total sialic acid, a significant proximal-to-distal increase in Neu5Gc was detected. In all cases, a good correlation between the amount of Neu5Gc, the activity of the hydroxylase and the level of hydroxylase mRNA was observed. However, Western-blot analysis revealed considerable accumulation of less active enzyme in the post partum period, which persisted until adulthood. No evidence for cytosolic factors influencing the hydroxylase activity or for the formation of truncated enzyme was found, raising the possibility that other regulatory mechanisms are involved. The relevance of these results in the formation of Neu5Gc as a receptor for certain pig enteric pathogens is also discussed.
Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A
Authors: SÁNCHEZ-TORRES Paloma , VISSER Jaap , BENEN Jacques A.E.
Published: February 2003

Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed β-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689] and the modelled enzyme—substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 8762—8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154→Arg and Asp154→Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme—substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the pKa of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186—Asp221 pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689]. Substitution of Asp186 and Asp221 by Asn186 and Asn221 was expected to stabilize the enzyme. However, the Asp186→Asn/Asp221→Asn enzyme appeared less stable than the wild-type enzyme, even at pH6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp186—Asp221 pair.
Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform
Authors: SHEPHERD Malcolm , McSORLEY Theresa , OLSEN Aileen E. , JOHNSTON Lee Ann , THOMSON Neil C. , BAILLIE George S. , HOUSLAY Miles D. , BOLGER Graeme B.
Published: February 2003

We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of ‘long’ PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein—PDE4B4 chimaera. PDE4B4 exhibited a Km for cAMP of 5.4μM and a Vmax, relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone} with an IC50 of 83nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.
Hypoxic activation of the atrial natriuretic peptide gene promoter through direct and indirect actions of hypoxia-inducible factor-1
Authors: CHUN Yang-Sook , HYUN Ju-Yeon , KWAK Yong-Geun , KIM In-San , KIM Chan-Hyung , CHOI Eunjoo , KIM Myung-Suk , PARK Jong-Wan
Published: February 2003

Atrial natriuretic peptide (ANP) is a cardiac peptide, the transcription of which is up-regulated in the ischaemic ventricle. However, the molecular mechanism of ANP induction is unclear. This study demonstrated that ANP mRNA expression in rat ventricular myocardium is induced in an early phase of ischaemia, preceded by hypoxia-inducible factor-1 (HIF-1) α expression. The ANP gene was also induced by hypoxia or HIF-1 inducers such as CoCl2 and desferrioxamine in H9c2 and neonatal cardiomyocytes. The 2307bp 5′-flanking region of the rat ANP gene was cloned and fused to the luciferase gene. Evidence of the promoter activity was only apparent in the myocytes and was induced by hypoxia and HIF-1 inducers. The overexpression of HIF-1α markedly enhanced ANP promoter activity, and a dominant-negative isoform completely suppressed it. We demonstrated that the promoter regions are essential for hypoxic ANP induction. One promoter region, containing the HIF-1-binding sequence, is regulated directly by HIF-1. The other region is also activated by HIF-1 despite having no HIF-1-binding sequence. These results suggest that HIF-1 enhances the transactivation of the ANP gene in hypoxic myocytes, implying that stimulation of the ANP promoter by HIF-1 may in fact be responsible for the induction of the ANP gene in ischaemic ventricular myocardium.
Nitric oxide co-ordinates the activities of the capacitative and non-capacitative Ca2+-entry pathways regulated by vasopressin
Authors: MONEER Zahid , DYER Jeanette L. , TAYLOR Colin W.
Published: February 2003

In A7r5 vascular smooth muscle cells vasopressin, via arachidonic acid, regulates two Ca2+-entry pathways. Capacitative Ca2+ entry (CCE), activated by empty Ca2+ stores, is inhibited by arachidonic acid, and non-capacitative Ca2+ entry (NCCE) is stimulated by it. This reciprocal regulation ensures that all Ca2+ entry is via NCCE in the presence of vasopressin, while CCE mediates a transient Ca2+ entry only after removal of vasopressin. We demonstrate that type III NO synthase (NOS III) is expressed in A7r5 cells and that NO inhibits CCE. Inhibition of CCE by vasopressin requires NOS III and the requirement lies downstream of arachidonic acid. Activation of soluble guanylate cyclase by NO and subsequent activation of protein kinase G are required for inhibition of CCE. Stimulation of NCCE by vasopressin also requires NOS III, but the stimulation is neither mimicked by cGMP nor blocked by inhibitors of soluble guanylate cyclase or protein kinase G. We conclude that arachidonic acid formed in response to vasopressin stimulates NOS III. NO then directly stimulates Ca2+ entry through NCCE and, via protein kinase G, it inhibits CCE. The additional amplification provided by the involvement of guanylate cyclase and protein kinase G ensures that CCE will always be inhibited when vasopressin activates NCCE.
Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase
Authors: IYENGAR Akshai , BEARNE Stephen L.
Published: February 2003

CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH3 or l-glutamine as the nitrogen source. GTP is required as an allosteric effector to promote glutamine hydrolysis. In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (residues 102—118) within the synthase domain of Escherichia coli CTP synthase. Mutant K102A CTP synthase exhibited wild-type activity with respect to NH3 and glutamine; however, the R105A, D107A, L109A and G110A enzymes exhibited wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation. The E103A, R104A and H118A enzymes exhibited no glutamine-dependent activity and were only partially active with NH3. Although these observations were compatible with impaired activation by GTP, the apparent affinity of the D107A, L109A and G110A enzymes for GTP was reduced only 2—4-fold, suggesting that these residues do not play a significant role in GTP binding. In the presence of GTP, the kcat values for glutamine hydrolysis by the D107A and L109A enzymes were identical with that of wild-type CTP synthase. Overall, the kinetic properties of L109A CTP synthase were consistent with an uncoupling of glutamine hydrolysis from CTP formation that occurs because an NH3 tunnel has its normal structure altered or fails to form. L109F CTP synthase was prepared to block totally the putative NH3 tunnel; however, this enzyme's rate of glutamine-dependent CTP formation and glutaminase activity were both impaired. In addition, we observed that mutation of amino acids located between residues 102 and 118 in the synthase domain can affect the enzyme's glutaminase activity, suggesting that these residues interact with residues in the glutamine amide transfer domain because they are in close proximity or via a conformationally dependent signalling mechanism.
Analyses of temporal regulatory elements of the prosaposin gene in transgenic mice
Authors: SUN Ying , WITTE David P. , JIN Peng , GRABOWSKI Gregory A.
Published: February 2003

The expression of prosaposin is temporally and spatially regulated at transcriptional and post-translational levels. Transgenic mice with various 5′-flanking deletions of the prosaposin promoter fused to luciferase (LUC) reporters were used to define its temporal regulatory region. LUC expression in the transgenic mice carrying constructs with 234bp (234LUC), 310bp (310LUC) or 2400bp (2400LUC) of the 5′-flanking region was analysed in the central nervous system and eye throughout development. For 310LUC and 2400LUC, low-level LUC activity was maintained until embryonal day 18 in brain, eye and spinal cord. The peak level of LUC activity was at birth, with return to a plateau (1/3 of peak) throughout adulthood. Deletion of the region that included the retinoic acid-receptor-related orphan receptor (RORα)-binding site and sequence-specific transcription factor (Sp1) cluster sites (44—310bp) suppressed the peak of activity. By comparison, the peak level for 234LUC was shifted 2 weeks into neonatal life in the brain, but not in the eye, and no peak of activity was observed in the spinal cord. The endogenous prosaposin mRNA in eye, spinal cord and cerebellum had low-level expression before birth and continued to increase into adulthood. In cerebrum, the endogenous mRNA showed similar expression profile to constructs 310LUC, 2400LUC and 234LUC, with the peak expression at 1 week and a decreased level in adult. In the brain of the newborn, 2400LUC was highly expressed in the trigeminal ganglion and brain stem regions when compared with the generalized expression pattern for endogenous prosaposin mRNA. These results suggest that the modifiers (RORα- and Sp1-binding sites) residing within 310bp of the 5′-flanking region mediate developmental regulation in the central nervous system and eye. Additional regulatory elements outside the 5′ region of the 2400bp promoter fragment appear to be essential for the physiological control of the prosaposin locus.
Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells
Authors: HII Charles S.T. , COSTABILE Maurizio , MAYNE George C. , DER Channing J. , MURRAY Andrew W. , FERRANTE Antonio
Published: February 2003

The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKCβI, ∊, θ and ζ. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.