bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of the structure–activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis
Authors: LEMAITRE Nadine , CALLEBAUT Isabelle , FRENOIS Frédéric , JARLIER Vincent , SOUGAKOFF Wladimir
Published: August 2015

In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (⩽ 0.004unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06–0.25unit/mg) and one (T167L) exhibited a wild-type activity (1.51units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 Å sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an α-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.
Haem oxygenase-1: a novel player in cutaneous wound repair and psoriasis?
Authors: HANSELMANN Christine , MAUCH Cornelia , WERNER Sabine
Published: August 2015

Haem oxygenase (HO) is the rate-limiting enzyme in the degradation of haem. In addition to its obvious role in iron metabolism, a series of findings indicate an important role for HO in cellular protection against oxidative stress. This effect might be of particular importance during wound healing and also in inflammatory disease. Therefore we determined the expression of the two HO isoenzymes, HO-1 and HO-2, during the healing process of full-thickness excisional wounds in mice. We show a remarkable induction of HO-1 mRNA and protein expression within three days after skin injury. After completion of wound healing, HO-1 expression declined to basal levels. By contrast, expression of HO-2 was not significantly modulated by skin injury. In situ hybridization and immunohistochemistry revealed high HO-1 expression in inflammatory cells of the granulation tissue and in keratinocytes of the hyperproliferative epithelium. A strong overexpression of HO-1 was also observed in the skin of patients suffering from the inflammatory skin disease psoriasis. In addition, HO-2 mRNA levels were increased in the skin of psoriatic patients. Similar to wounded skin, inflammatory cells and keratinocytes of the hyperthickened epidermis were the major producers of HO-1 in psoriatic skin. In vitro studies with cultured keratinocytes revealed a potential role for reactive oxygen species (ROS), but not for growth factors and pro-inflammatory cytokines, as inducers of HO-1 expression in inflamed skin. Our findings suggest a novel role for HO in wound healing and inflammatory skin disease, where it might be involved in haem degradation and in the protection of cells from the toxic effects of ROS.
Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd1 nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation
Authors: WILSON Emma K. , BELLELLI Andrea , CUTRUZZOLÀ Francesca , ZUMFT Walter G. , GUTIERREZ Aldo , SCRUTTON Nigel S.
Published: August 2015

cd 1 nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd1 nitrite reductase of Pseudomonas aeruginosahave established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzerienzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosacd1 nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr10 stabilizing a water molecule co-ordinated to the d1-haem. Given the intimate association of the N-terminal extension with the d1-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzericd1 nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzericd1 nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzericd1 nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosain the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd1 nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzericd1 nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.
The Escherichia coli CcmG protein fulfils a specific role in cytochrome c assembly
Authors: REID Eleanor , COLE Jeff , EAVES Deborah J.
Published: August 2015

In Escherichia coli K-12, c-type cytochromes are synthesized only during anaerobic growth with trimethylamine-N-oxide, nitrite or low concentrations of nitrate as the terminal electron acceptor. A thioredoxin-like protein, CcmG, is one of 12 proteins required for their assembly in the periplasm. Its postulated function is to reduce disulphide bonds formed between correctly paired cysteine residues in the cytochrome c apoproteins prior to haem attachment by CcmF and CcmH. We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming proteins, DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic acid. An anti-CcmG polyclonal antibody was used in Western-blot analysis to probe the redox state of CcmG in mutants defective in the synthesis of other proteins essential for cytochrome c assembly. The oxidized form of CcmG accumulated not only in trxA or dipZ mutants defective in the transfer of electrons from the cytoplasm for disulphide isomerization and reduction reactions in the periplasm, but also in ccmF and ccmH mutants. The requirement of both CcmF and CcmH for the reduction of the disulphide bond in CcmG indicates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer from the membrane-associated DipZ (also known as DsbD) to CcmH. The data support a model proposed by others in which CcmG catalyses one of the last reactions specific to cytochrome c assembly.
Differential regulation of expression of genes encoding uncoupling proteins 2 and 3 in brown adipose tissue during lactation in mice
Authors: PEDRAZA Neus , SOLANES Gemma , IGLESIAS Roser , VÁZQUEZ Manuel , GIRALT Marta , VILLARROYA Francesc
Published: August 2015

Thermogenic activity in brown adipose tissue (BAT) decreases during lactation; the down-regulation of the gene encoding uncoupling protein 1 (UCP1) is involved in this process. Our studies show that UCP2 mRNA expression does not change during the breeding cycle in mice. In contrast, UCP3 mRNA is down-regulated in lactation but it recovers after weaning, in parallel with UCP1 mRNA. This leads to a decrease in the content of UCP3 in BAT mitochondria during lactation. Lowering the energy-sparing necessities of lactating dams by decreasing litter size or feeding with a high-fat diet prevented the down-regulation of UCP1 mRNA and UCP3 mRNA. In most cases this resulted in a less marked decrease in UCP1 and UCP3 protein in BAT mitochondria owing to lactation. Fasting for 24h caused a different response in UCP1 and UCP3 mRNA expression: it decreased UCP1 mRNA levels but had no effect on UCP3 mRNA abundance in virgin mice; it even increased UCP3 mRNA expression in lactating dams. These changes did not lead to modifications in UCP1 or UCP3 protein abundance. Whereas acute treatment with peroxisome-proliferator-activated receptor (PPAR)α and PPARγ agonists increased UCP1 mRNA levels only in lactating dams, UCP3 mRNA expression was induced by both kinds of PPAR activator in lactating dams and by PPARα agonists in virgin mice. It is concluded that modifications of UCP2 mRNA levels are not part of the physiological adaptations taking place in BAT during lactation. In contrast, the down-regulation of UCP3 mRNA expression and mitochondrial UCP3 content is consistent with a role for the gene encoding UCP3 in the decrease in metabolic fuel oxidation and thermogenesis in BAT during lactation.
Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins
Authors: GILLOOLY David J. , SIMONSEN Anne , STENMARK Harald
Published: August 2015

PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling.
Telomerase is regulated by protein kinase C-ζ in human nasopharyngeal cancer cells
Authors: YU Cheng-Chou , LO Shi-Ching , WANG Tzu-Chien V.
Published: August 2015

Telomerase, a specialized ribonucleoprotein reverse transcriptase that directs the synthesis of telomeric DNA, is repressed in normal human somatic cells, but is activated in most cancers. Little is known concerning how telomerase activity is activated and maintained in cancer cells. We have shown previously that inhibition of protein kinase C (PKC) decreases the telomerase activity of human nasopharyngeal carcinoma (NPC) cells. Here, we provide evidence that the decrease of telomerase activity by PKC inhibition is not mediated by transcriptional down-regulation of hTERT, the catalytic protein of human telomerase. In vitro phosphorylation studies revealed that exogenous addition of PKC-α, -βI, -δ or -ζ led to restoration of telomerase activity in the crude extracts of PKC-inhibited NPC cells. However, depletion of PKC-α and -βI in vivo had no detectable effect on the telomerase activity of NPC cells. Using antisense oligonucleotides against individual PKC isotypes, we observed that telomerase activity was inhibited only by the antisense oligonucleotide against PKC-ζ but not by those against PKC-α, -βI or -δ. Taken together, these data demonstrate that PKC participates in the regulation of telomerase activity by direct or indirect phosphorylation of telomerase proteins, and that PKC-ζ is the PKC isotype that functions in vivo in the NPC cells.
Plasmin-dependent elimination of the growth-factor-like domain in urokinase causes its rapid cellular uptake and degradation
Authors: POLIAKOV Alexei , TKACHUK Vsevolod , OVCHINNIKOVA Tatyana , POTAPENKO Natalia , BAGRYANTSEV Svyatoslav , STEPANOVA Victoria
Published: August 2015

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) act in concert to mediate pericellular proteolysis and to stimulate intracellular signalling responsible for cell migration and proliferation. uPA is composed of three domains, a proteolytic domain (PD), a kringle domain (KD) and a growth-factor-like domain (GFD), the last of which mediates the interaction with uPAR. We demonstrate that uPA, associated with the surface of U937 cells, undergoes plasmin-mediated cleavage of the Lys46-Ser47 bond with elimination of the GFD. Using recombinant forms of uPA, we show that a uPA variant lacking the GFD (r-uPA∆GFD) and unable to associate with uPAR is rapidly cleared from the cell surface. Binding and internalization of r-uPA∆GFD are markedly decreased in the presence of 39kDa receptor-associated protein (RAP), the antagonist of several endocytic receptors of the low-density lipoprotein receptor family, suggesting that this protein clearance pathway is used for r-uPA∆GFD. In contrast with rapidly internalized r-uPA∆GFD, the intact recombinant single-chain urokinase with wild-type structure (r-uPAwt) bound to uPAR is retained on the cell surface. Soluble uPAR protects uPA from cleavage by plasmin that results in the elimination of GFD, suggesting that uPAR might protect cell-bound urokinase from plasmin-mediated cleavage between the GFD and KD and subsequent degradation.
Phosphate-binding loop and Rab GTPase function: mutations at Ser29 and Ala30 of Rab5 lead to loss-of-function as well as gain-of-function phenotype
Authors: LI Guangpu , LIANG Zhimin
Published: August 2015

Ras-like GTPases contain a structurally conserved GTP-binding domain. An important element of the GTP-binding domain is the phosphate-binding loop, which contains two Gly residues (Gly12 and Gly13) in Ras. Because the two Gly residues are crucial for normal Ras function, it is intriguing that they are not conserved in other Ras-like GTPases, including the Rab GTPases; for example, the equivalent residues in Rab5 are Ser29 and Ala30. The present study builds on earlier biochemical characterizations of the Rab5 mutants containing substitutions at Ala30 and provides a comprehensive analysis of the structure–function relationship of the Rab5 phosphate-binding loop. We have generated 19 new mutants containing amino acid substitutions at Ser29 and determined whether these Ser29 mutants, as well as the Ala30 mutants, remain able to stimulate the endocytosis of horseradish peroxidase in baby hamster kidney cells. A total of 11 mutants lose the activity of stimulating endocytosis. Of these 11 mutants, 9 are defective in membrane association. In contrast, 27 mutants remain able to stimulate endocytosis. Five of them induce a novel cellular phenotype: cell rounding and detachment from culture dishes. They also induce super-large early endosomes such as the constitutively activated Rab5:Q79L mutant. Biochemical results suggest that the constitutive activation of Rab5 requires an increased nucleotide exchange rate and/or decreased GTPase activity. This study establishes functional significance for the phosphate-binding loop of Rab5 and shows that mutations in this region lead to either a loss-of-function or a gain-of-function phenotype, indicating a structure–function relationship distinct from that of Ras.
Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin
Authors: BOLDT Henning B. , OVERGAARD Michael T. , LAURSEN Lisbeth S. , WEYER Kathrin , SOTTRUP-JENSEN Lars , OXVIG Claus
Published: August 2015

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.
Sda-antigen-like structures carried on core 3 are prominent features of glycans from the mucin of normal human descending colon
Authors: CAPON Calliope , MAES Emmanuel , MICHALSKI Jean-Claude , LEFFLER Hakon , KIM Young S.
Published: August 2015

This paper describes structural characterization by NMR, MS and degradative studies of mucin glycans from normal human descending colon obtained freshly at autopsy. The saccharides were mainly based on core 3 (GlcNAcβ1-3GalNAc). Among the terminal saccharide determinants Sda/Cad-antigen-like structures were prominent, and Lewis x, sialyl Lewis x and sulphated Lewis x were found as minor components, whereas blood group H and A antigenic determinants were absent. The saccharides were markedly different from those of mucins from colon cancers or colon cancer cell lines analysed so far, in which cores 1 and 2 are prominent features, and in which various other terminal determinants have been found, but not Sda/Cad.
RGS18 is a myeloerythroid lineage-specific regulator of G-protein-signalling molecule highly expressed in megakaryocytes
Authors: YOWE David , WEICH Nadine , PRABHUDAS Mercy , POISSON Louis , ERRADA Patrick , KAPELLER Rosanna , YU Kan , FARON Laura , SHEN Minhui , CLEARY Jennifer , WILKIE Thomas M. , GUTIERREZ-RAMOS Carlos , HODGE Martin R.
Published: August 2015

Myelopoiesis and lymphopoiesis are controlled by haematopoietic growth factors, including cytokines, and chemokines that bind to G-protein-coupled receptors (GPCRs). Regulators of G-protein signalling (RGSs) are a protein family that can act as GTPase-activating proteins for Gαi- and Gαq-class proteins. We have identified a new member of the R4 subfamily of RGS proteins, RGS18. RGS18 contains clusters of hydrophobic and basic residues, which are characteristic of an amphipathic helix within its first 33 amino acids. RGS18 mRNA was most highly abundant in megakaryocytes, and was also detected specifically in haematopoietic progenitor and myeloerythroid lineage cells. RGS18 mRNA was not detected in cells of the lymphoid lineage. RGS18 was also highly expressed in mouse embryonic 15-day livers, livers being the principal organ for haematopoiesis at this stage of fetal development. RGS1, RGS2 and RGS16, other members of the R4 subfamily, were expressed in distinct progenitor and mature myeloerythroid and lymphoid lineage blood cells. RGS18 was shown to interact specifically with the Gαi-3 subunit in membranes from K562 cells. Furthermore, overexpression of RGS18 inhibited mitogen-activated-protein kinase activation in HEK-293/chemokine receptor 2 cells treated with monocyte chemotactic protein-1. In yeast cells, RGS18 overexpression complemented a pheromone-sensitive phenotype caused by mutations in the endogeneous yeast RGS gene, SST2. These data demonstrated that RGS18 was expressed most highly in megakaryocytes, and can modulate GPCR pathways in both mammalian and yeast cells in vitro. Hence RGS18 might have an important role in the regulation of megakaryocyte differentiation and chemotaxis.
Portable sulphotransferase domain determines sequence specificity of heparan sulphate 3-O-sulphotransferases
Authors: YABE Tomio , SHUKLA Deepak , SPEAR Patricia G. , ROSENBERG Robert D. , SEEBERGER Peter H. , SHWORAK Nicholas W.
Published: August 2015

3-O-Sulphates are the rarest substituent of heparan sulphate and are therefore ideally suited to the selective regulation of biological activities. Individual isoforms of heparan sulphate d-glucosaminyl 3-O-sulphotransferase (3-OST) exhibit sequence-specific action, which creates heparan sulphate structures with distinct biological functions. For example, 3-OST-1 preferentially generates binding sites for anti-thrombin, whereas 3-OST-3 isoforms create binding sites for the gD envelope protein of herpes simplex virus 1 (HSV-1), which enables viral entry. 3-OST enzymes comprise a presumptive sulphotransferase domain and a divergent N-terminal region. To localize determinants of sequence specificity, we conducted domain swaps between cDNA species. The N-terminal region of 3-OST-1 was fused with the sulphotransferase domain of 3-OST-3A to generate N1-ST3A. Similarly, the N-terminal region of 3-OST-3A was fused to the sulphotransferase domain of 3-OST-1 to generate N3A-ST1. Wild-type and chimaeric enzymes were transiently expressed in COS-7 cells and extracts were analysed for selective generation of binding sites for anti-thrombin. 3-OST-1 was 270-fold more efficient at forming anti-thrombin-binding sites than 3-OST-3A, indicating its significantly greater selectivity for substrates that can be 3-O-sulphated to yield such sites. N3A-ST1 was as active as 3-OST-1, whereas the activity of N1-ST3A was as low as that of 3-OST-3A. Analysis of Chinese hamster ovary cell transfectants revealed that only 3-OST-3A and N1-ST3A generated gD-binding sites and conveyed susceptibility to infection by HSV-1. Thus sequence-specific properties of 3-OSTs are defined by a self-contained sulphotransferase domain and are not directly influenced by the divergent N-terminal region.
Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75
Authors: PROVOST Patrick , DOUCET Johanne , STOCK Alexander , GERISCH Günther , SAMUELSSON Bengt , RÅDMARK Olof
Published: August 2015

Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys75 of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys75 is essential for this interaction.
Characterization and regulation of inositol monophosphatase activity in Mycobacterium smegmatis
Authors: NIGOU Jérôme , BESRA Gurdyal S.
Published: August 2015

Mycobacterium tuberculosis and related members of the genus Mycobacterium contain a number of inositol-based lipids, such as phosphatidylinositol mannosides, lipomannan and lipoarabinomannan. The synthesis of phosphatidylinositol in M. smegmatis is essential for growth and myo-inositol is a key metabolite for mycobacteria. Little is known about the biosynthesis of inositol in mycobacteria and the only known de novo pathway for myo-inositol biosynthesis involves a two-step process. First, cyclization of glucose 6-phosphate to afford myo-inositol 1-phosphate via inositol-1-phosphate synthase and, secondly, dephosphorylation of myo-inositol 1-phosphate by inositol monophosphatase (IMP) to afford myo-inositol. The following report examines IMP activity in M. smegmatis extracts, with regard to pH dependence, bivalent cation re quirement, univalent cation inhibition, regulation by growth and carbon source. We show that IMP activity, which is optimal at the end of the exponential growth phase in Sauton's medium, is Mg2+-dependent. Moreover, IMP activity is inhibited by Li+ and Na+, with Li+ also being able to inhibit growth of M. smegmatis in vivo. This study represents a first step in the delineation of myo-inositol biosynthesis in mycobacteria.
Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases
Authors: OZ Iris , AVIDAN Orna , HIZI Amnon
Published: August 2015

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3′-end processing and 3′-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.
A novel Ca2+-induced Ca2+ release mechanism mediated by neither inositol trisphosphate nor ryanodine receptors
Authors: WISSING Frank , NEROU Edmund P. , TAYLOR Colin W.
Published: August 2015

Members of both major families of intracellular Ca2+ channels, ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, are stimulated by substantial increases in cytosolic free Ca2+ concentration ([Ca2+]c). They thereby mediate Ca2+-induced Ca2+ release (CICR), which allows amplification and regenerative propagation of intracellular Ca2+ signals. In permeabilized hepatocytes, increasing [Ca2+]c to 10μM stimulated release of 30±1% of the intracellular stores within 60s; the EC50 occurred with a free [Ca2+] of 170±29nM. This CICR was abolished at 2°C. The same fraction of the stores was released by CICR before and after depletion of the IP3-sensitive stores, and CICR was not blocked by antagonists of IP3 receptors. Ryanodine, Ruthenium Red and tetracaine affected neither the Ca2+ content of the stores nor the CICR response. Sr2+ and Ba2+ (EC50 = 166nM and 28μM respectively) mimicked the effects of increased [Ca2+] on the intracellular stores, but Ni2+ blocked the passive leak of Ca2+ without blocking CICR. In rapid superfusion experiments, maximal concentrations of IP3 or Ca2+ stimulated Ca2+ release within 80ms. The response to IP3 was complete within 2s, but CICR continued for tens of seconds despite a slow [half-time (t1/2) = 3.54±0.07s] partial inactivation. CICR reversed rapidly (t1/2 = 529±17ms) and completely when the [Ca2+] was reduced. We conclude that hepatocytes express a novel temperature-sensitive, ATP-independent CICR mechanism that is reversibly activated by modest increases in [Ca2+], and does not require IP3 or ryanodine receptors or reversal of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase. This mechanism may both regulate the Ca2+ content of the intracellular stores of unstimulated cells and allow even small intracellular Ca2+ signals to be amplified by CICR.
Regulation of phosphatidylcholine and phosphatidylethanolamine synthesis in rat hepatocytes by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)
Authors: HOUWELING Martin , KLEIN Wil , GEELEN Math J. H.
Published: August 2015

The present study was undertaken to study the role of AMP-activated kinase (AMPK) in the biosynthesis of two major membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of AMPK, produced dose-dependent inhibition of the incorporation of [3H]choline and [3H]ethanolamine into PC and PE, respectively. Determination of the cellular uptake of choline and ethanolamine showed that the reduced synthesis of PC and PE did not result from impaired uptake of these two precursors. The decreased synthesis of PC was not mirrored by a reduction in the activities of the enzymes of the CDP-choline pathway. The diminution of PE biosynthesis, however, was paralleled by a depressed activity of CTP:phosphoethanolamine cytidylyltransferase (ET), the pace-setting enzyme of the CDP-ethanolamine pathway. AICAR treatment of hepatocytes stimulated the conversion of choline into betaine, indicating that reduced PC synthesis most probably resulted from a decrease in the availability of choline. In addition, AICAR induced a 50% reduction in the cellular level of diacylglycerols, which may further impair the synthesis of PC and PE. The results thus indicate that AICAR inhibits the biosynthesis of PC and PE and that the effect is exerted at different sites in the two pathways. Increased oxidation of choline to betaine is the main target of AICAR in the PC pathway, whereas inhibition of ET activity is the locus of AICAR action in the PE pathway.
Specific increase in leptin production in obese (falfa) rat adipose cells
Authors: TURBAN Sophie , HAINAULT Isabelle , TRUCCOLO Johan , ANDRE Jocelyne , FERRE Pascal , QUIGNARD-BOULANGE Annie , GUERRE-MILLO Michèle
Published: August 2015

In the obese state, enlarged adipose cells display an altered gene-expression profile and metabolic capacity. The aim of this study was to gain insight into their secretory function, by assessing two secreted proteins, leptin and angiotensinogen, in adipose cells of obese (fa/fa) Zucker rats. A marked and co-ordinate increase in leptin mRNA, gene transcription and promoter activity was observed in obese compared with lean (Fa/fa) rat adipose cells, and this resulted in increased leptin release in culture. Two sets of observations suggest that this effect is due to the fa mutation. First, adipose-cell leptin release was higher in heterozygous (Fa/fa) than in homozygous (Fa/Fa) lean rats. Second, leptin release was not enhanced in enlarged adipose cells of FalFa rats fed a high-fat diet for 15 days. At variance with leptin, angiotensinogen production was not significantly increased in the obese cells. Dexamethasone stimulated both leptin and angiotensinogen release in lean and obese rat adipose cells. The magnitude of leptin stimulation was higher in fa/fa than in Fa/fa rats, whereas angiotensinogen release was increased to the same extent in both genotypes. These observations suggest that leptin production is specifically enhanced in enlarged adipose cells of obese Zucker rats and that cell hypertrophy is not the sole determinant of this feature. Increased leptin production might be related to disruption of leptin signalling by the fa mutation.
myo-Inositol hexakisphosphate is a major component of an extracellular structure in the parasitic cestode Echinococcus granulosus
Authors: IRIGOÍN Florencia , FERREIRA Fernando , FERNÁNDEZ Cecilia , SIM Robert B. , DÍAZ Alvaro
Published: August 2015

myo-Inositol hexakisphosphate (IP6) is an abundant intracellular component of animal cells. In this study we describe the presence of extracellular IP6 in the hydatid cyst wall (HCW) of the larval stage of the cestode parasite Echinococcus granulosus. The HCW comprises an inner cellular layer and an outer, acellular (laminated) layer up to 2mm in thickness that protects the parasite from host immune cells. A compound, subsequently identified as IP6, was detected in and purified from an HCW extract on the basis of its capacity to inhibit complement activation. The identification of the isolated compound was carried out by a combination of NMR, MS and TLC. The majority of IP6 in the HCW was found in the acellular layer, with only a small fraction of the compound being extracted from cells. In the laminated layer, IP6 was present in association with calcium, and accounted for up to 15% of the total dry mass of the HCW. IP6 was not detected in any other structures or stages of the parasite. Our results imply that IP6 is secreted by the larval stage of the parasite in a polarized fashion towards the interface with the host. This is the first report of the secretion of IP6, and the possible implications beyond the biology of E. granulosus are discussed.
Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors
Authors: MEACCI Elisabetta , CENCETTI Francesca , FORMIGLI Lucia , SQUECCO Roberta , DONATI Chiara , TIRIBILLI Bruno , QUERCIOLI Franco , ORLANDINI Sandra ZECCHI , FRANCINI Fabio , BRUNI Paola
Published: August 2015

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C2C12 myoblasts SPP elicited significant Ca2+ mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca2+ response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca2+ transients performed with a spectrophotofluorimeter showed that the rise in Ca2+ was strictly dependent on availability of extracellular Ca2+. Cell treatment with pertussis toxin partially prevented the Ca2+ response induced by SPP, indicating that Gi-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca2+ mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca2+, although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca2+ increase, supporting the notion that Ca2+ mobilization in C2C12 cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.
Effect of brefeldin A on heparan sulphate biosynthesis in Madin–Darby canine kidney cells
Authors: KOLSET Svein Olav , PRYDZ Kristian , FJELDSTAD Katja , SAFAIYAN Fariba , VUONG Tram Thu , GOTTFRIDSSON Eva , SALMIVIRTA Markku
Published: August 2015

Brefeldin A (BFA) perturbs the organization of the Golgi apparatus, such that Golgi stack components are fused with the endoplasmic reticulum (ER) and separated from the trans-Golgi network. In many cell types, BFA blocks the secretion of macromolecules but still allows the action of Golgi enzymes in the ER. Treatment of cells with BFA has been reported to inhibit the secretion of heparan sulphate (HS) proteoglycans and alter the structure of their HS components, but the nature of such structural alterations has not been characterized in detail. We analysed the effect of BFA on HS biosynthesis in Madin—Darby canine kidney (MDCK) cells, in which the Golgi complex is more resistant towards BFA than in most other cell types. We found that MDCK cells were able to secrete HS proteoglycans in spite of BFA treatment. However, the secretion of HS was reduced and the secreted HS differed from that produced by untreated cells. In BFA-treated cells, two structurally distinct pools of HS were generated. One pool was similar to HS from control cells, with the exception that the 6-O-sulphation of glucosamine (GlcN) residues was reduced. In contrast, the other pool consisted of largely unmodified N-acetylheparosan polymers with a low (<20%) proportion of N-sulphated GlcN residues but a substantial proportion of N-unsubstituted GlcN units, indicating that it had been acted upon by N-deacetylases and partly by the N-sulphotransferases, but not by O-sulphotransferases. Together, these findings represent a previously unrecognized alteration in HS biosynthesis caused by BFA, and differ dramatically from our previous findings in MDCK cells pertaining to the undersulphation of HS caused by sodium chlorate treatment.
The glucose-6-phosphatase system
Authors: van SCHAFTINGEN Emile , GERIN Isabelle
Published: August 2015

Glucose-6-phosphatase (G6Pase), an enzyme found mainly in the liver and the kidneys, plays the important role of providing glucose during starvation. Unlike most phosphatases acting on water-soluble compounds, it is a membrane-bound enzyme, being associated with the endoplasmic reticulum. In 1975, W. Arion and co-workers proposed a model according to which G6Pase was thought to be a rather unspecific phosphatase, with its catalytic site oriented towards the lumen of the endoplasmic reticulum [Arion, Wallin, Lange and Ballas (1975) Mol. Cell. Biochem. 6, 75–83]. Substrate would be provided to this enzyme by a translocase that is specific for glucose 6-phosphate, thereby accounting for the specificity of the phosphatase for glucose 6-phosphate in intact microsomes. Distinct transporters would allow inorganic phosphate and glucose to leave the vesicles. At variance with this substrate-transport model, other models propose that conformational changes play an important role in the properties of G6Pase. The last 10 years have witnessed important progress in our knowledge of the glucose 6-phosphate hydrolysis system. The genes encoding G6Pase and the glucose 6-phosphate translocase have been cloned and shown to be mutated in glycogen storage disease type Ia and type Ib respectively. The gene encoding a G6Pase-related protein, expressed specifically in pancreatic islets, has also been cloned. Specific potent inhibitors of G6Pase and of the glucose 6-phosphate translocase have been synthesized or isolated from micro-organisms. These as well as other findings support the model initially proposed by Arion. Much progress has also been made with regard to the regulation of the expression of G6Pase by insulin, glucocorticoids, cAMP and glucose.
Activation of bacterial ceramidase by anionic glycerophospholipids: possible involvement in ceramide hydrolysis on atopic skin by Pseudomonas ceramidase
Authors: KITA Katsuhiro , SUEYOSHI Noriyuki , OKINO Nozomu , INAGAKI Masanori , ISHIDA Hideharu , KISO Makoto , IMAYAMA Shuhei , NAKAMURA Takashi , ITO Makoto
Published: August 2015

We have reported previously that the ceramidase from Pseudomonas aeruginosa AN17 isolated from a patient with atopic dermatitis requires detergents for hydrolysis of ceramide (Cer) [Okino, Tani, Imayama and Ito (1998) J. Biol. Chem. 273, 14368–14373]. In the present study, we report that some glycerophospholipids strongly activated the hydrolysis of Cer by Pseudomonas ceramidase in the absence of detergents. Among the glycerophospholipids tested, cardiolipin was most effective in stimulating hydrolysis of Cer followed by phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, whereas phosphatidylcholine, lysophosphatidic acid and diacylglycerol were less effective. Interestingly, Staphylococcus aureus-derived lipids, which contain cardiolipin and phosphatidylglycerol as major lipid components, also strongly enhanced the hydrolysis of normal Cer, as well as the human skin-specific ω-hydroxyacyl Cer, by the enzyme in the absence of detergents. It was confirmed that several strains of P. aeruginosa, including AN17, secrete a significant amount of staphylolytic proteases to lyse S. aureus cells, resulting in the release of cardiolipin and phosphatidylglycerol. Since both P. aeruginosa and S. aureus are suspected of being present in microflora of atopic skin, we speculate that S. aureus-derived glycerophospholipids stimulate the hydrolysis of Cer in atopic skin by bacterial ceramidase.
Decorin suppresses transforming growth factor-β-induced expression of plasminogen activator inhibitor-1 in human mesangial cells through a mechanism that involves Ca2+-dependent phosphorylation of Smad2 at serine-240
Authors: ABDEL-WAHAB Nadia , WICKS Stephen J. , MASON Roger M. , CHANTRY Andrew
Published: August 2015

Transforming growth factor-β (TGFβ) is a key mediator of extracellular matrix (ECM) accumulation in sclerotic kidney diseases such as diabetic nephropathy. One of the main target cells for TGFβ in the kidney are glomerular mesangial cells, which respond by increasing expression of ECM proteins, such as collagens, laminin and fibronectin, while suppressing the expression of ECM-degrading proteases and increasing the synthesis of ECM protease inhibitors, including plasminogen activator inhibitor-1. Previous studies have shown that exposure of mesangial cells to chronic high-glucose conditions, such as those seen in diabetes, increases ECM deposition in a mechanism involving glucose-mediated up-regulation of TGFβ expression. Naturally occurring inhibitors of this TGFβ-dependent fibrotic response include decorin, a small leucine-rich proteoglycan. While the mechanism by which TGFβ stimulates gene expression via the Smad signal-transduction pathway is becoming clear, the precise mechanism by which decorin may impinge upon TGFβ activity remains to be established. In this study, for the first time we provide evidence that decorin can disrupt glucose- and TGFβ/Smad-dependent transcriptional events in human mesangial cells through a mechanism that involves an increase in Ca2+ signalling, the activation of Ca2+/calmodulin-dependent protein kinase II and ensuing phosphorylation of Smad2 at Ser-240. We show that decorin also induces Ser-240 phospho-Smad hetero-oligomerization with Smad4 and the nuclear localization of this complex independently of TGFβ receptor activation. Thus, in human mesangial cells, the mechanism of decorin-mediated inhibition of TGFβ signalling may involve activation of Ca2+ signalling, the subsequent phosphorylation of Smad2 at a key regulatory site, and the sequestration of Smad4 in the nucleus.
Mesangial cell gelatinase A synthesis is attenuated by oscillating hyperbaric pressure
Authors: EN-NIA Abdelaziz , REISDORFF Julia , STEFANIDIS Ioannis , FLOEGE Juergen , HEINRICH Peter C. , MERTENS Peter R.
Published: August 2015

Glomerular hypertension has been established as a major factor contributing to glomerular scarring. Underlying cellular mechanisms leading to matrix accumulation are largely unknown. The isolated effect of oscillating hyperbaric pressure [OP; Pmax 50mmHg (1mmHg = 0.133kPa), Pmean 24mmHg, with a fixed oscillation of 60/min] on matrix-degrading protease secretion by rat mesangial cells (MCs) was analysed using a pressure chamber model described previously [Mertens, Espenkott, Venjakob, Heintz, Handt and Sieberth (1998) Hypertension 32, 945–952]. MCs were grown under atmospheric pressure (AP) or a controlled OP, and protease synthesis and gene transcription were analysed. A distinct biphasic cellular response to OP with stimulated gelatinase A protein expression and enzyme activity during the initial 24h, and subsequent inhibition, was apparent, as shown by gelatin zymography. Gelatinase B activity remained unchanged. The abundance of gelatinase A transcripts, determined by reverse transcriptase-PCR, indicated a concordant regulation of gene transcription. To elucidate underlying regu latory events, reporter constructs were transfected. In these experiments, a recently identified response element, RE-1, conferred a significant stimulatory effect within the initial 4h of OP. Nuclear protein/RE-1 binding studies revealed additional complexes from 5min up to 3h after OP exposure, with intensities dependent on Pmax. STAT3 was identified as a component of these novel complexes. Down-regulation of cis-activity after 48h of OP exposure was not transferred via the proximal 1686bp of the gelatinase A regulatory sequence. In conclusion, hyperbaric OP elicits time-dependent changes in rat MC gelatinase A gene transcription.
Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R
Authors: van CALSTEREN Marie-Rose , PAU-ROBLOT Corinne , BÉGIN André , ROY Denis
Published: August 2015

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillusrhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical 1H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; d-glucose, 2; d-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: {3Rhaα-3Glcβ-3[Gal4,6(R)Pyα-2]Rhaα-3Rhaα-3Rhaα-2Glcα-}n where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete 1H and 13C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.