bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B
Authors: CORMONT Mireille , GAUTIER Nadine , ILC Karine , Le MARCHAND-BRUSTEL Yannick
Published: August 2015

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.
α1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins
Authors: LÖSTER Klemens , VOSSMEYER Dörte , HOFMANN Werner , REUTTER Werner , DANKER Kerstin
Published: August 2015

Integrins are heterodimeric adhesion receptors consisting of α- and β-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein–protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic α1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the α1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on α1β1-specific substrates. The α1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, α-actinin and pp125FAK. It seems to account for α1-specific signalling, since when peptides that mimic the cytoplasmic domain of α1 are transferred into cells, they influence α1β1-specific adhesion, presumably by competing for binding partners. For α1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.
Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine
Authors: VOSHOL Peter J. , SCHWARZ Margrit , RIGOTTI Attilio , KRIEGER Monty , GROEN Albert K. , KUIPERS Folkert
Published: August 2015

Scavenger receptor class B, type I (SR-BI) is expressed in the intestines of rodents and has been suggested to be involved in the absorption of dietary cholesterol. The aim of this study was to determine whether intestinal SR-BI expression is affected in animal models with altered bile delivery to the intestine and impaired cholesterol absorption. SR-BI protein and mRNA levels were determined in proximal and distal small intestine from control, bile-duct-ligated and bile-diverted rats and from control and bile-duct-ligated mice. Two genetically altered mouse models were studied: multidrug resistance-2 P-glycoprotein-deficient [Mdr2(−/−)] mice that produce phospholipid/cholesterol-free bile, and cholesterol 7α-hydroxylase-deficient [Cyp7a(−/−)] mice, which exhibit qualitative and quantitative changes in the bile-salt pool. Cholesterol-absorption efficiency was quantified using a dual-isotope ratio method. SR-BI was present at the apical membrane of enterocytes in control rats and mice and was more abundant in proximal than in distal segments of the intestine. In bile-duct-ligated animals, levels of SR-BI protein were virtually absent and mRNA levels were decreased by ≈ 50%. Bile-diverted rats, Mdr2(−/−) mice and Cyp7a(−/−) mice showed decreased levels of intestinal SR-BI protein while mRNA levels were unaffected. Cholesterol absorption was reduced by > 90% in bile-duct-ligated and bile-diverted animals and in Cyp7a(−/−) mice, whereas Mdr2(−/−) mice showed an ≈ 50% reduction. This study shows that SR-BI is expressed at the apical membrane of enterocytes of rats and mice, mainly in the upper intestine where cholesterol absorption is greatest, and indicates that bile components play a role in post-transcriptional regulation of SR-BI expression. Factors associated with cholestasis appear to be involved in transcriptional control of intestinal SR-BI expression. The role of SR-BI in the cholesterol-absorption process remains to be defined.
Heat shock stimulation of a tilapia heat shock protein 70 promoter is mediated by a distal element
Authors: MOLINA Alfredo , Di MARTINO Emmanuel , MARTIAL Joseph A. , MULLER Marc
Published: August 2015

We reported previously that a tilapia (Oreochromis mossambicus) heat shock protein 70 (HSP70) promoter is able to confer heat shock response on a reporter gene after transient expression both in cell culture and in microinjected zebrafish embryos. Here we present the first functional analysis of a fish HSP70 promoter, the tiHSP70 promoter. Using transient expression experiments in carp EPC (epithelioma papulosum cyprini) cells and in microinjected zebrafish embryos, we show that a distal heat shock response element (HSE1) at approx. −800 is predominantly responsible for the heat shock response of the tiHSP70 promoter. This element specifically binds an inducible transcription factor, most probably heat shock factor, and a constitutive factor. The constitutive complex is not observed with the non-functional, proximal HSE3 sequence, suggesting that both factors are required for the heat shock response mediated by HSE1.
A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern
Authors: PRIOR Alison , JONES John T. , BLOK Vivian C. , BEAUCHAMP Jeremy , McDERMOTT Lindsay , COOPER Alan , KENNEDY Malcolm W.
Published: August 2015

Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-d,l-α-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C11 to C24, with optimal binding at C15. Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.
Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione
Authors: CHENG Hailing , TCHAIKOVSKAYA Tatyana , TU Yen-Sheng L. , CHAPMAN Jason , QIAN Biao , CHING Wei-Mei , TIEN Ming , ROWE Jonathan D. , PATSKOVSKY Yury V. , LISTOWSKY Irving , TU Chen-Pei D.
Published: August 2015

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (kcat/Km) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50–180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix α4 region of rGSTM4-4 account for this pKa difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6–10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.
An integrated study of threonine-pathway enzyme kinetics in Escherichia coli
Authors: CHASSAGNOLE Christophe , RAÏS Badr , QUENTIN Eric , FELL David A. , MAZAT Jean-Pierre
Published: August 2015

We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.
Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases and phosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis
Authors: SCHUBERT Kathryn M. , DURONIO Vincent
Published: August 2015

Alterations in the expression of various Bcl-2 family members may act as one means by which a cell's survival may be regulated. The mechanism by which cytokines regulate expression of Bcl-2 family members was examined in the haemopoietic cell line TF-1. Cytokine-induced Mcl-1 protein expression was shown to be controlled through a pathway dependent upon phosphatidylinositol 3-kinase (PI 3-kinase). The cytokine-induced increase in mRNA transcription was not dependent upon PI 3-kinase, thus dissociating the immediate-early transcription factors responsible for Mcl-1 transcription from the PI 3-kinase signalling pathway. In contrast, Mcl-1 mRNA levels were dependent upon MEK [mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase kinase] activation, suggesting a role for the Ras/MEK/MAPK pathway in Mcl-1 transcription. Activation of PI 3-kinase was shown to be necessary to stimulate Mcl-1 protein translation. This was not due to any effect on prolonging the half-life of the protein. Finally, the lipid second messenger ceramide was shown to cause a reduction in Mcl-1 protein translation, probably via its ability to inhibit protein kinase B activation, providing further clues regarding the death-inducing effect of this lipid.
Bile acid hydrophobicity is correlated with induction of apoptosis and/or growth arrest in HCT116 cells
Authors: POWELL Ashley A. , LaRUE Janna M. , BATTA A. K. , MARTINEZ Jesse D.
Published: August 2015

Faecal bile acids have long been associated with colon cancer; highly hydrophobic bile acids, which induce apoptosis, have been implicated in the promotion of colon tumours. The moderately hydrophobic chemopreventive agent ursodeoxycholic acid (UDCA) does not induce apoptosis; rather, it causes colon-derived tumour cells to arrest their growth. To investigate the relationship between bile acid hydrophobicity and biological activity we examined 26 bile acids for their capacity to induce apoptosis or alter cell growth. We found that the rapidity with which, and the degree to which, bile acids could induce apoptosis or growth arrest was correlated with their relative hydrophobicities. Of the bile acids tested, only deoxycholic acid (DCA) and chenodeoxycholic acid, the most hydrophobic bile acids tested, could induce apoptosis in less than 12h in the human colon cancer cell line HCT116. The moderately hydrophobic bile acids hyoDCA, lagoDCA, norDCA, homoUDCA and isoUDCA induced growth arrest at 12h but longer incubations resulted in apoptosis. Conjugation of glycine or taurine to the bile acids decreased relative hydrophobicity and eliminated biological activity in our assays. In addition, we tested a subset of these bile acids for their ability to translocate across cell membranes. When 14C-labelled and 3H-labelled DCA, UDCA and lagoDCA were added to cell cultures, we found only minimal uptake by colon cells, whereas hepatocytes had considerably higher absorption. These experiments suggest that hydrophobicity is an important determinant of the biological activity exhibited by bile acids but that under our conditions these activities are not correlated with cellular uptake.
Novel barnacle underwater adhesive protein is a charged amino acid-rich protein constituted by a Cys-rich repetitive sequence
Authors: KAMINO Kei
Published: August 2015

Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed.
Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival
Authors: JEONG Jinsook , HAN Innoc , LIM Yangmi , KIM Jungyean , PARK Ilseon , WOODS Anne , COUCHMAN John R. , OH Eok-Soo
Published: August 2015

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.
Modifications of eukaryotic initiation factor 4F (eIF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on eIF4F activity and total protein synthesis rates
Authors: SAGHIR Atif N. , TUXWORTH William J. , HAGEDORN Curt H. , McDERMOTT Paul J.
Published: August 2015

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F (eIF4F) activity are correlated with accelerated rates of total protein synthesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load, cardiocytes were electrically stimulated to contract (2Hz, 5ms pulses). Non-stimulated cardiocytes were used as controls. Adenovirus-mediated overexpression of wild-type eIF4E increased the total eIF4E pool by 120–140% above endogenous levels after 24h and produced a corresponding increase in eIF4F content. However, it did not accelerate total protein synthesis rates in quiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreased binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increased the quantity of eIF4F but the rate of total protein synthesis was decreased in quiescent and contracting cardiocytes. Overexpression of a mutant that blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity of eIF4F without any significant effect on total protein synthesis rates in quiescent or contracting cardiocytes. Overexpression of the eIF4E kinase Mnk-1 increased eIF4E phosphorylation without a corresponding increase in eIF4F complex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via adenoviral gene transfer; (2) the cap binding affinity of eIF4F is a rate-limiting determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4F alone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.
Identification of a novel neuroligin in humans which binds to PSD-95 and has a widespread expression
Authors: BOLLIGER Marc F. , FREI Karl , WINTERHALTER Kaspar H. , GLOOR Sergio M.
Published: August 2015

Neuroligins, first discovered in rat brain, form a family of three synaptically enriched membrane proteins. Using reverse transcription–PCR of human brain polyadenylated RNA and extensive database searches, we identified the human homologues of the three rat neuroligins and a cDNA encoding a fourth member, which we named neuroligin 4. Neuroligin 4has 63–73% amino acid identity with the other members of the human neuroligin family, and the same predicted domain structure. DNA database analyses, furthermore, indicated that a possible fifth neuroligin gene may be present in the human genome. Northern-blot analysis revealed expression of neuroligin 4 in heart, liver, skeletal muscle and pancreas, but barely at all in brain. Overexpression of neuroligin 4 cDNA in COS-7 cells led to the production of a 110kDa protein. Immunofluorescence analysis demonstrated that the protein was integrated into the plasma membrane. Overexpression of cDNAs encoding neuroligin 4 and the PDZ-domain protein, PSD-95, in COS-7 cells resulted in the formation of detergent-resistant complexes. Neuroligin 4 did not bind to ZO-1, another PDZ-domain protein. Together, our data show that the human neuroligin family is composed of at least one additional member, and suggest that neuroligin 4 may also be produced outside the central nervous system.
Importance of the 3′ untranslated region of ornithine decarboxylase mRNA in the translational regulation of the enzyme
Authors: LÖVKVIST WALLSTRÖM Eva , TAKAO Koichi , WENDT Anna , VARGIU Cristina , YIN Hong , PERSSON Lo
Published: August 2015

Translational regulation of ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of polyamines, appears to be an important mechanism in the strong feedback control as well as in the hypotonic induction of the enzyme. However, the exact mechanisms are not yet understood. The ODC mRNA has long 5′ and 3′ untranslated regions (UTRs) which may be involved in the translational control of the enzyme. In the present study we have used a series of stable transfectants of Chinese Hamster ovary cells expressing ODC mRNAs with various truncations in the 5′ and 3′ UTRs to investigate the importance of these regions. It is demonstrated that neither the 5′ UTR nor the 3′ UTR appears to be involved in the polyamine-mediated feedback control of ODC synthesis. The hypotonic induction of ODC, on the other hand, was shown to be highly dependent on the presence of the 3′ UTR, but not on the 5′ UTR, of ODC mRNA. Cells expressing ODC mRNAs lacking the 3′ UTR showed no, or only a very slight, induction of ODC whether the 5′ UTR was present or not, whereas the cell lines expressing ODC mRNAs containing the 3′ UTR (with or without the 5′ UTR) markedly induced ODC after a hypotonic shock. The present finding of a role for the ODC mRNA 3′ UTR in the hypotonic induction of ODC is the first demonstration of a specific effect of the 3′ UTR in the regulation of ODC.
Intracellular energetic units in red muscle cells
Authors: SAKS Valdur A. , KAAMBRE Tuuli , SIKK Peeter , EIMRE Margus , ORLOVA Ehte , PAJU Kalju , PIIRSOO Andres , APPAIX Florence , KAY Laurence , REGITZ-ZAGROSEK Vera , FLECK Eckart , SEPPET Enn
Published: August 2015

The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70–80% of maximal rate of respiration was achieved at ADP concentration below 20μM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20–40units/ml) and phosphoenolpyruvate (PEP; 5mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20–40%. Creatine (20mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50–500nM for 5min) decreased the apparent Km for exogenous ADP from 300–350μM to 50–60μM, increased inhibition of respiration by PK+PEP system up to 70–80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent Km for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes (‘intracellular energetic units’) with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.
Emergence during development of the white-adipocyte cell phenotype is independent of the brown-adipocyte cell phenotype
Authors: MOULIN Karine , TRUEL Nathalie , ANDRÉ Mireille , ARNAULD Emmanuelle , NIBBELINK Maryse , COUSIN Béatrice , DANI Christian , PÉNICAUD Luc , CASTEILLA Louis
Published: August 2015

In mammals, two types of adipose tissue are present, brown and white. They develop sequentially, as brown fat occurs during late gestation whereas white fat grows mainly after birth. However, both tissues have been shown to have great plasticity. Thus an apparent transformation of brown fat into white fat takes place during post-natal development. This observation raises questions about a possible conversion of brown into white adipocytes during development, although indirect data argue against this hypothesis. To investigate such questions in vivo, we generated two types of transgenic line. The first carried a transgene expressing Cre recombinase specifically in brown adipocytes under the control of the rat UCP1 promoter. The second corresponded to an inactive lacZ gene under the control of the human cytomegalovirus promoter. This dormant gene is inducible by Cre because it contains a Stop sequence between two loxP sequences, separating the promoter from the coding sequence. Adipose tissues of progeny derived by crossing independent lines established from both constructs were investigated. LacZ mRNA corresponding to the activated reporter gene was easily detected in brown fat and not typically in white fat, even by reverse transcriptase PCR experiments. These data represent the first direct experimental proof that, during normal development, most white adipocytes do not derive from brown adipocytes.
Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum
Authors: SWEADNER Kathleen J. , DONNET Claudia
Published: August 2015

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca2+-ATPase) has recently been determined at 2.6 Å (note 1 Å = 0.1nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647–655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca2+-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca2+-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.
A role of tensin in skeletal-muscle regeneration
Authors: ISHII Akiko , LO S. Hao
Published: August 2015

Regeneration of skeletal muscle requires the activation, proliferation, differentiation and fusion of satellite cells to generate new muscle fibres. This study was designed to determine the role of tensin in this process. Cardiotoxin was used to induce regeneration in the anterior tibial muscles of tensin-knockout and wild-type mice. From histological analysis, we found that the regeneration process lasted longer in knockout than in wild-type mice. To investigate the mechanism involved in this delay, we examined each regeneration step in animals and cultured primary cells. We found fewer proliferating myogenic cells identified by bromodeoxyuridine and desmin double labelling in knockout mice on the first 2 days after injury. Expression of myosin, paxillin, dystrophin and dystrophin-associated proteins were delayed in knockout mice. Withdrawal from the cell cycle was less efficient in isolated knockout myoblasts, and the fusion capacity was reduced in these cells as well. These defects in regeneration most likely contributed to the 9-fold increase of centrally nucleated fibres occurring in the non-injected knockout mice. Our results demonstrated clearly that tensin plays a role in skeletal-muscle regeneration.
Reducible cationic lipids for gene transfer
Authors: WETZER Barbara , BYK Gerardo , FREDERIC Marc , AIRIAU Marc , BLANCHE Francis , PITARD Bruno , SCHERMAN Daniel
Published: August 2015

One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA–disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA–lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA–lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA–lipid complexes after intracellular reduction and represent a tool for improved vectorization.
Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors
Authors: LUCIANI Nathalie , de ROCQUIGNY Hugues , Turcaud Serge , ROMIEU Anthony , ROQUES Bernard P.
Published: August 2015

The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of ‘intramolecularly quenched fluorescence’ which may fulfill this aim. One, S1 [Pya21-Nop22-bET-1(19–35)], is the (19–35) fragment of the natural peptide big-ET-1(1–38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S2) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH2, from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya21-Nop22-bET-1(19–35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors.
Transcriptional activities of p73 splicing variants are regulated by inter-variant association
Authors: UEDA Yoshihide , HIJIKATA Makoto , TAKAGI Shinji , CHIBA Tsutomu , SHIMOTOHNO Kunitada
Published: August 2015

p73 has been identified as a gene that encodes a protein with significant identity with the tumour suppressor p53. The main structural difference between p73 and p53 is the additional C-terminal region of p73. Six isoforms of p73 with differing C-terminal structures, α, β, γ, δ, ∊ and ξ, have been reported. These variants differ in transcriptional activity on p53-responsive promoters. Here we report a possible mechanism of transcriptional activation by p73 splicing variants. C-terminal deletion mutants of p73α showed a significantly higher level of transcriptional activity than wild-type p73α, suggesting that the C-terminal structure of p73α functions to repress the transcriptional activity of p73α. The results of immunoprecipitation assays and two-hybrid assays in mammalian cells showed that the p73 variants interacted with each other, but not with p53. The transcriptional activity of p73β was reduced by co-expression with either p73α or p73∊, which bears an identical C-terminal structure to p73α. Co-expression of the C-terminal portion of p73α or p73∊ with p73β also resulted in reduced transcriptional activity. Moreover, we observed that the level of endogenous p21 protein induced by p73β was decreased by co-expression of full-length p73∊ or the C-terminal region of p73α or p73∊. These observations suggest that p73-mediated gene expression is regulated by the interactions of p73 splicing variants in the cell.
Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles
Authors: STUBENRAUCH Kay , GLEITER Stefan , BRINKMANN Ulrich , RUDOLPH Rainer , LILIE Hauke
Published: August 2015

The development of cell-type-specific delivery systems is highly desirable for gene-therapeutic applications. Current virus-based vector systems show broad cell specificity, which results in the need to restrict the natural tropism of these viral systems. Here we demonstrate that tumour-cell-specific virus-like particles can be functionally assembled in vitro from recombinant viral coat protein expressed in Escherichia coli. The insertion of a negatively charged peptide in the HI loop of polyoma VP1 interferes with the binding of VP1 to the natural recognition site on mammalian cells and also serves as an adapter for the coupling of antibody fragments that contain complementary charged fusion peptides. A recombinant antibody fragment of the tumour-specific anti-(Lewis Y) antibody B3 could be coupled to the mutant VP1 by engineered polyionic peptides and an additional disulphide bond. With this system an entirely recombinant cell-specific delivery system assembled in vitro could be generated that transfers genes preferentially to cells presenting the tumour-specific antigen on the cell surface.
Identification of a new subfamily of sulphotransferases: cloning and characterization of canine SULT1D1
Authors: TSOI Carrie , FALANY Charles N. , MORGENSTERN Ralf , SWEDMARK Stellan
Published: August 2015

Sulphation is an important conjugation pathway in drug metabolism that has been studied in several species including humans. However, few studies have been performed using the dog as a subject. In this report we describe the cloning and characterization of a canine cytosolic sulphotransferase (SULT). The overall primary structure of this enzyme is very similar to that of a rat phenol-sulphating enzyme found in the EMBL Database and to a mouse SULT termed amine-N-sulphotransferase (81% identity). The expressed canine SULT conjugates small phenols and aromatic amines such as dopamine, minoxidil, p-nitrophenol and 5-hydroxytryptamine, but not dehydroepiandrosterone or β-oestradiol. These results are in agreement with the results reported for the mouse SULT. In contrast with the mouse enzyme, the canine SULT does not conjugate eicosanoid compounds, i.e. prostaglandins, thromboxane B2 or leukotriene E4. The canine SULT is expressed at high levels in the colon of both genders; it is also expressed in the small intestine, kidney and liver. Furthermore, because the canine, mouse and rat SULT forms exhibit significant sequence identity (more than 80%), they seem to represent a distinct group in the SULT family tree. This suggestion is strengthened by the low identity with other SULTs. The subfamily that is most similar to this new group is SULT1A, with approx. 60% similarity. However, the mouse and canine enzymes are not characterized by the efficient sulphation of p-nitrophenol, dopamine, β-oestradiol or oestrone. Thus these results seem to exclude them from the SULT1A subfamily. We therefore propose a new subfamily in the phenol SULT family, designated SULT1D, and consequently the canine enzyme is termed SULT1D1.
Characterization of Sp17: a ubiquitous three domain protein that binds heparin
Authors: WEN Ying , RICHARDSON Richard T. , WIDGREN Esther E. , O'RAND Michael G.
Published: August 2015

Sp17 is a protein that was originally thought to be expressed exclusively in the testis and whose primary function was binding to the extracellular matrix of the oocyte. Several recent reports have implicated Sp17 as having a role in cell–cell adhesion and/or cell migration in transformed, lymphocytic and haematopoietic cells, possibly through its interaction with extracellular heparan sulphate. In the present study, we report that Sp17's central domain (amino acids 61–117), spanning exon 3, is critical for heparin binding. Sp17has two additional functional domains, an N-terminal domain similar to the dimer-interaction site in the cAMP-dependent protein kinase IIα regulatory subunit and a C-terminal calmodulin-binding domain. The mouse gene for Sp17 is 6.5kb and contains four exons. Although Sp17 expression is highest in the testis, it is present in all of the mouse somatic tissues examined and is highly conserved throughout all mammalian species. Sp17's central domain, which is necessary for heparin binding, exhibits the greatest sequence divergence of all three domains. The Sp17 gene is induced in metastatic cells and during mucosal immune responses, and the protein appears to play an important role in cell migration and/or adhesion in somatic cells, as well as in male germ cells.
Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding
Authors: KRAMER Barbara , FERRARI David M. , KLAPPA Peter , PÖHLMANN Nicole , SÖLING Hans-Dieter
Published: August 2015

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b′ domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as ‘scrambled’ ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a′ domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b′ domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.
Isolation and characterization of the human stearoyl-CoA desaturase gene promoter: requirement of a conserved CCAAT cis-element
Authors: ZHANG Lin , GE Lan , TRAN Tai , STENN Kurt , PROUTY Stephen M.
Published: August 2015

Stearoyl-CoA desaturase is the rate-limiting enzyme in the production of mono-unsaturated fatty acids. We have recently cloned and characterized the human Scd cDNA and SCD (the stearoyl-CoA desaturase structural gene) on chromosome 10, as well as the non-transcribed pseudogene on chromosome 17. In order to further define SCD regulation and function, we have isolated and characterized the promoter of the structural gene. Screening of chromosome-10-specific libraries resulted in the isolation of 4.1kb of SCD sequence upstream of the translation start site. Binding sites for transcription factors critical for mouse Scd1 and Scd2 promoter activity, such as sterol-regulated-element-binding protein and nuclear factor Y, were present in the human SCD promoter (Scd is the mouse stearoyl-CoA desaturase gene). Deletion analysis in HaCaT keratinocytes identified a critical region for promoter activity between nts 496–609 upstream of the translation start site. Site-directed mutagenesis of binding sites in this region identified the CCAAT box as the critical cis-element for SCD promoter activity. An electrophoretic mobility-shift assay confirmed that this element binds nuclear proteins from HaCaT keratinocytes. The polyunsaturated-fatty-acid (PUFA) response element, previously identified in the promoters of mouse Scd1 and Scd2, was found to be conserved in the human SCD promoter, and contained the critical CCAAT cis-element. A minimal promoter construct including this region was responsive to fatty acids, with oleate and linoleate decreasing transcription and stearate increasing it. These studies indicate that CCAAT-box-binding proteins activate SCD transcription in cultured keratinocytes and that fatty acids modulate transcription, most likely through the conserved PUFA response element.
Ferritin and the response to oxidative stress
Authors: ORINO Kouichi , LEHMAN Lori , TSUJI Yoshiaki , AYAKI Hitoshi , TORTI Suzy V. , TORTI Frank M.
Published: August 2015

Iron is required for normal cell growth and proliferation. However, excess iron is potentially harmful, as it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton chemistry. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels. Chief among these is the sequestration of iron in ferritin. Ferritin is a 24 subunit protein composed of two subunit types, termed H and L. The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. In the present study we report that increased synthesis of both subunits of ferritin occurs in HeLa cells exposed to oxidative stress. An increase in the activity of iron responsive element binding proteins in response to oxidative stress was also observed. However, this activation was transient, allowing ferritin protein induction to subsequently proceed. To assess whether ferritin induction reduced the accumulation of ROS, and to test the relative contribution of ferritin H and L subunits in this process, we prepared stable transfectants that overexpressed either ferritin H or ferritin L cDNA under control of a tetracycline-responsive promoter. We observed that overexpression of either ferritin H or ferritin L reduced the accumulation of ROS in response to oxidant challenge.
Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data
Authors: CORTÉS Antoni , CASCANTE Marta , CÁRDENAS María Luz , CORNISH-BOWDEN Athel
Published: August 2015

The concentration of an inhibitor that decreases the rate of an enzyme-catalysed reaction by 50%, symbolized i0.5, is often used in pharmacological studies to characterize inhibitors. It can be estimated from the common inhibition plots used in biochemistry by means of the fact that the extrapolated inhibitor concentration at which the rate becomes infinite is equal to −i0.5. This method is, in principle, more accurate than comparing the rates at various different inhibitor concentrations, and inferring the value of i0.5 by interpolation. Its reciprocal, 1/i0.5, is linearly dependent on v0/V, the uninhibited rate divided by the limiting rate, and the extrapolated value of v0/V at which 1/i0.5 is zero allows the type of inhibition to be characterized: this value is 1 if the inhibition is strictly competitive; greater than 1 if the inhibition is mixed with a predominantly competitive component; infinite (i.e. 1/i0.5 does not vary with v0/V) if the inhibition is pure non-competitive (i.e. mixed with competitive and uncompetitive components equal); negative if the inhibition is mixed with a predominantly uncompetitive component; and zero if it is strictly uncompetitive. The type of analysis proposed has been tested experimentally by examining inhibition of lactate dehydrogenase by oxalate (an uncompetitive inhibitor with respect to pyruvate) and oxamate (a competitive inhibitor with respect to pyruvate), and of cytosolic malate dehydrogenase by hydroxymalonate (a mixed inhibitor with respect to oxaloacetate). In all cases there is excellent agreement between theory and experiment.