bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel steroid receptor co-activator protein (SRAP) as an alternative form of steroid receptor RNA-activator gene: expression in prostate cancer cells and enhancement of androgen receptor activity
Authors: KAWASHIMA Hidenori , TAKANO Haruna , SUGITA Syozo , TAKAHARA Yuki , SUGIMURA Kazunobu , NAKATANI Tatsuya
Published: December 2002

We have cloned a cDNA coding for a novel steroid receptor co-activator protein termed SRAP from a rat prostate library. Although the nucleotide sequence of the SRAP has 78.2% identity to that of the human steroid receptor RNA activator (SRA), a novel RNA molecule which was reported to act as an RNA transcript without being translated into protein [Lanz, McKenna, Onate, Albrecht, Wong, Tsai, Tsai and O'Malley (1999) Cell 97, 17—27], the cDNA of SRAP is capable of generating a functional protein. Glutathione S-transferase pull-down assays showed that SRAP associates with the partial androgen receptor (AR) protein composed of a DNA-binding domain and an activation function 2. Luciferase assays demonstrated that SRAP enhances the transactivation activity of the AR, the glucocorticoid receptor and the peroxisome proliferator-activated receptor γ1 in a ligand-dependent manner. Using a green fluorescent protein (GFP) fusion-protein construct, we demonstrated in vivo translation of the GFP-SRAP fusion protein in HeLa cells co-transfected with pSG5AR and reporter gene in the presence of 5α-dihydrotestosterone (DHT). Co-transfection of the GFP-SRAP fusion protein expression plasmid enhanced the transactivation activity of AR whereas incorporation of mutations in SRAP of the fusion protein resulted in loss of enhancement of the transactivation activity. Northern blot analysis and reverse transcriptase PCR assays showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and the human prostate cancer cells line DU-145, co-transfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by the anti-androgen flutamide, but the transactivation activities still remained high even in the presence of 5μM flutamide, suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer.
Crystal structure of nitrous oxide reductase from Paracoccus denitrificans at 1.6 A resolution
Authors: HALTIA Tuomas , BROWN Kieron , TEGONI Mariella , CAMBILLAU Christian , SARASTE Matti , MATTILA Kimmo , DJINOVIC-CARUGO Kristina
Published: December 2002

N2O is generated by denitrifying bacteria as a product of NO reduction. In denitrification, N2O is metabolized further by the enzyme N2O reductase (N2OR), a multicopper protein which converts N2O into dinitrogen and water. The structure of N2OR remained unknown until the recent elucidation of the structure of the enzyme isolated from Pseudomonas nautica. In the present paper, we report the crystal structure of a blue form of the enzyme that was purified under aerobic conditions from Paracoccus denitrificans. N2OR is a head-to-tail homodimer stabilized by a multitude of interactions including two calcium sites located at the intermonomeric surface. Each monomer is composed of two domains: a C-terminal cupredoxin domain that carries the dinuclear electron entry site known as CuA, and an N-terminal seven-bladed β-propeller domain which hosts the active-site centre CuZ. The electrons are transferred from CuA to CuZ across the subunit interface. CuZ is a tetranuclear copper cluster in which the four copper ions (Cu1 to Cu4) are ligated by seven histidine imidazoles, a hydroxyl or water oxygen and a bridging inorganic sulphide. A bound chloride ion near the CuZ active site shares one of the ligand imidazoles of Cu1. This arrangement probably influences the redox potential of Cu1 so that this copper is stabilized in the cupric state. The treatment of N2OR with H2O2 or cyanide causes the disappearance of the optical band at 640nm, attributed to the CuZ centre. The crystal structure of the enzyme soaked with H2O2 or cyanide suggests that an average of one copper of the CuZ cluster has been lost. The lowest occupancy is observed for Cu3 and Cu4. A docking experiment suggests that N2O binds between Cu1 and Cu4 so that the oxygen of N2O replaces the oxygen ligand of Cu4. Certain ligand imidazoles of Cu1 and Cu2, as well as of Cu4, are located at the dimer interface. Particularly those of Cu2 and Cu4 are parts of a bonding network which couples these coppers to the CuA centre in the neighbouring monomer. This structure may provide an efficient electron transfer path for reduction of the bound N2O.
Triadimefon, a fungicidal triazole-type P450 inhibitor, induces brassinosteroid deficiency-like phenotypes in plants and binds to DWF4 protein in the brassinosteroid biosynthesis pathway
Authors: ASAMI Tadao , MIZUTANI Masaharu , SHIMADA Yukihisa , GODA Hideki , KITAHATA Nobutaka , SEKIMATA Katsuhiko , HAN Sun-Young , FUJIOKA Shozo , TAKATSUTO Suguru , SAKATA Kanzo , YOSHIDA Shigeo
Published: December 2002

Triadimefon (Bayleton®), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14α-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the phenotype was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.
DNA recognition by the androgen receptor: evidence for an alternative DNA-dependent dimerization, and an active role of sequences flanking the response element on transactivation
Authors: HAELENS Annemie , VERRIJDT Guy , CALLEWAERT Leen , CHRISTIAENS Valerie , SCHAUWAERS Kris , PEETERS Ben , ROMBAUTS Wilfried , CLAESSENS Frank
Published: December 2002

The androgen receptor has a subset of target DNA sequences, which are not recognized by any other steroid receptors. The androgen selectivity of these sequences was proposed to be the consequence of the ability of the androgen receptor to dimerize on direct repeats of 5′-TGTTCT-3′-like sequences. This is in contrast with the classical non-selective elements consisting of inverted repeats of the 5′-TGTTCT-3′ elements separated by three nucleotides and which are recognized by other steroid receptors in addition to the androgen receptor. We demonstrate that while the DNA-binding domain of the oestrogen receptor is unable to dimerize on direct repeats, dimeric binding can be rescued by replacing the second Zn finger and part of the hinge region by the corresponding fragment of the androgen receptor, but not the glucocorticoid receptor. In this study, we investigate the androgen receptor binding to all natural androgen-selective response elements described so far. We show that a 12-amino acid C-terminal extension of the DNA-binding domain is required for high-affinity binding of the androgen receptor to all these elements. For one androgen-specific low-affinity binding site, the flanking sequences do not contribute to the invitro affinity of the androgen receptor DNA-binding domain. Surprisingly, however, they control the transcriptional activity of the androgen receptor in transient transfection experiments. In conclusion, we give evidence that the alternative DNA-dependent dimerization of the androgen receptor on direct repeats is a general mechanism for androgen specificity in which the second Zn finger and hinge region are involved. In addition, the sequences flanking an androgen-response element can control the activity of the androgen receptor.
The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1alpha, Jak1 and Jak2
Authors: OSAKI Makoto , TAN Lujian , CHOY Bob K. , YOSHIDA Yasuhiro , CHEAH Kathryn S.E. , AURON Philip E. , GOLDRING Mary B.
Published: December 2002

Interferon-γ (IFN-γ) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene (COL2A1) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-γ and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-γ inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-γ suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning −6368 to +125bp in the absence and presence of the intronic enhancer and stimulated activity of the γ-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1α-binding activity in nuclear extracts. These responses to IFN-γ were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1α Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-γ had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (γ2A)- and Stat1α (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-γ was rescued by overexpression of Stat1α, but not by either Stat1α Y701F or Stat1β. Functional analysis using deletion constructs showed that the IFN-γ response was retained in the COL2A1 core promoter region spanning −45 to +11bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-γ persisted in the presence of multiple deletions within the −45/+11bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-γ requires Jak1, Jak2 and Stat1α and suggest that this response involves indirect interaction of activated Stat1α with the general transcriptional machinery that drives constitutive COL2A1 expression.
Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle
Authors: NIIRO Naohisa , KOGA Yasuhiko , IKEBE Mitsuo
Published: December 2002

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both α-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.
Structural elucidation of zwitterionic carbohydrates derived from glycosphingolipids of the porcine parasitic nematode Ascaris suum
Authors: FRIEDL Claudia H. , LOCHNIT Günter , ZÄHRINGER Ulrich , BAHR Ute , GEYER Rudolf
Published: December 2002

Carbohydrates substituted with phosphocholine (PC) and phosphoethanolamine (PE) were released from zwitterionic glycosphingolipids of the pig parasitic nematode Ascaris suum by treatment with endoglycoceramidase. Individual glycans were obtained by HPLC on porous graphitic carbon followed by high-pH anion-exchange chromatography. In addition to the known pentasaccharides Galα3GalNAcβ4[PC6]GlcNAcβ3Manβ4Glc and Galα3GalNAcβ4[PC6]GlcNAcβ3[PE6]Manβ4Glc, the corresponding tri- and tetra-saccharides, as well as components with elongated structures, could be identified by matrix-assisted laser-desorption ionization—time-of-flight MS, methylation analysis, 1H- and 13C-NMR spectroscopy, exoglycosidase cleavage and electrospray ionization ion-trap MS. The extended components comprised novel structural motifs such as di-substituted α-galactose carrying two β-linked galactosyl residues, which were found to bear, in part, further fucose, galactose, N-acetylgalactosamine and/or N-acetylglucosamine moieties. Furthermore, additional fucosylation of the PC-substituted N-acetylglucosamine and a non-terminal fucosyl motif were detected. In conclusion, this study contributes significant new information on the glycome of nematodes.
Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter
Authors: ZELCER Noam , SAEKI Tohru , BOT Ilse , KUIL Annemieke , BORST Piet
Published: December 2002

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect (Spodoptera frugiperda) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC5010μM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50100, 250 and 500μM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a Km and Vmax of 29±7μM and 660±63pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis.
Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3zeta
Authors: BIRKENFELD Jörg , BETZ Heinrich , ROTH Dagmar
Published: December 2002

Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3ζ isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3ζ. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3ζ inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3ζ-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1.
New enzyme belonging to the family of molybdenum-free nitrate reductases
Authors: ANTIPOV Alexey N. , SOROKIN Dimitry Y. , L'VOV Nikolay P. , KUENEN J. Gijs
Published: December 2002

A novel molybdenum-free nitrate reductase was isolated from the obligate chemolithoautotrophic and facultative anaerobic, (halo)alkaliphilic sulphur-oxidizing bacterium Thioalkalivibrio nitratireducens strain ALEN 2. The enzyme was found to contain vanadium and haem c as cofactors. Its native molecular mass was determined as 195kDa, and the enzyme consists of four identical subunits with apparent molecular masses of 57kDa. Apart from nitrate, the enzyme can utilize nitrite, chlorate, bromate, selenate and sulphite as electron acceptors. Moreover, it also has a haloperoxidase activity.
Glucagon-like peptide 1 and fatty acids amplify pulsatile insulin secretion from perifused rat islets
Authors: CUNNINGHAM Barbara A. , RICHARD Ann-Marie T. , DILLON Joseph S. , DALEY Jennifer T. , CIVELEK Vildan N. , DEENEY Jude T. , YANEY Gordon C. , CORKEY Barbara E. , TORNHEIM Keith
Published: December 2002

Glucose-induced insulin secretion from isolated, perifused rat islets is pulsatile with a period of about 5—10min, similar to the insulin oscillations that are seen in healthy humans but which are impaired in Type II diabetes. We evaluated the pattern of enhancement by the potent incretin, glucagon-like peptide 1 (GLP-1). GLP-1 increased the amplitude of pulses and the magnitude of insulin secretion from the perifused islets, without affecting the average time interval between pulses. Forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine had the same effect, suggesting that the effect was due to elevated cAMP levels. The possibility that cAMP might enhance the amplitude of pulses by reducing phosphofructo-2-kinase (PFK-2) activity was eliminated when the liver isoform of PFK-2 was shown to be absent from β-cells. The possibility that cAMP enhanced pulsatile secretion, at least in part, by stimulating lipolysis was supported by the observations that added oleate had a similar effect on secretion, and that the incretin effect of GLP-1 was inhibited by the lipase inhibitor orlistat. These data show that the physiological incretin GLP-1 preserves and enhances normal pulsatile insulin secretion, which may be essential in proposed therapeutic uses of GLP-1 or its analogues.
Solution structure of a chemosensory protein from the moth Mamestra brassicae
Authors: MOSBAH Amor , CAMPANACCI Valérie , LARTIGUE Audrey , TEGONI Mariella , CAMBILLAU Christian , DARBON Hervé
Published: December 2002

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. A number of such proteins, of molecular mass 13kDa, have been isolated from different sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. CSPMbraA6, a 112-amino-acid antennal protein, has been expressed in a soluble form in large quantities in the Escherichi coli periplasm. NMR structure determination of CSPMbraA6 has been performed with 1H- and 15N-labelled samples. The calculated structures present an average root mean square deviation about the mean structure of 0.63Å for backbone atoms and 1.27Å for all non-hydrogen atoms except the 12 N-terminal residues. The protein is well folded from residue 12 to residue 110, and consists of a non-bundle α-helical structure with six helices connected by αα loops. It has a globular shape, with overall dimensions of 32Å×28Å×24Å. A channel is visible in the hydrophobic core, with dimensions of 3Å×9Å×21Å. In some of the 20 solution structures calculated, this channel is closed either by Trp-94 at one end or by Tyr-26 at the other end; in some other solutions, this channel is closed at both ends. Binding experiments with 12-bromododecanol indicate that the CSPMbraA6 structure is modified upon ligand binding.
A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells
Authors: PARTANEN Sanna , STORCH Stephan , LÖFFLER Hans-Gerhard , HASILIK Andrej , TYYNELÄ Jaana , BRAULKE Thomas
Published: December 2002

The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786—2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.
Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease
Authors: WANG Guirong , CHRISTENSEN Neil D. , WIGDAHL Brian , GUTTENTAG Susan H. , FLOROS Joanna
Published: December 2002

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42kDa) undergoes post-translational processing to generate an 8kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131→Ile) is associated with several pulmonary diseases. The Thr131→Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn129-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions.
Hepatocyte nuclear factor-4alpha mediates the stimulatory effect of peroxisome proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) on glucose-6-phosphatase catalytic subunit gene transcription in H4IIE cells
Authors: BOUSTEAD Jared N. , STADELMAIER Beth T. , EEDS Angela M. , WIEBE Peter O. , SVITEK Christina A. , OESER James K. , O'BRIEN Richard M.
Published: December 2002

It has recently been shown that adenoviral-mediated expression of peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1 α) in hepatocytes stimulates glucose-6-phosphatase catalytic subunit (G6Pase) gene expression. A combination of fusion gene, gel retardation and chromatin immunoprecipitation assays revealed that, in H4IIE cells, PGC-1α mediates this stimulation through an evolutionarily conserved region of the G6Pase promoter that binds hepatocyte nuclear factor-4α.
Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family
Authors: LORENZO Carmen , SALINAS Gustavo , BRUGNINI Andreina , WERNSTEDT Christer , HELLMAN Ulf , GONZÁLEZ-SAPIENZA Gualberto
Published: December 2002

Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38kDa subunits. Whereas the 22kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the Oγ atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.
Protein kinase CK2: structure, regulation and role in cellular decisions of life and death
Authors: LITCHFIELD David W.
Published: December 2002

Protein kinase CK2 ('casein kinase II') has traditionally been classified as a messenger-independent protein serine/threonine kinase that is typically found in tetrameric complexes consisting of two catalytic (α and/or α′) subunits and two regulatory β subunits. Accumulated biochemical and genetic evidence indicates that CK2 has a vast array of candidate physiological targets and participates in a complex series of cellular functions, including the maintenance of cell viability. This review summarizes current knowledge of the structural and enzymic features of CK2, and discusses advances that challenge traditional views of this enzyme. For example, the recent demonstrations that individual CK2 subunits exist outside tetrameric complexes and that CK2 displays dual-specificity kinase activity raises new prospects for the precise elucidation of its regulation and cellular functions. This review also discusses a number of the mechanisms that contribute to the regulation of CK2 in cells, and will highlight emerging insights into the role of CK2 in cellular decisions of life and death. In this latter respect, recent evidence suggests that CK2 can exert an anti-apoptotic role by protecting regulatory proteins from caspase-mediated degradation. The mechanistic basis of the observation that CK2 is essential for viability may reside in part in this ability to protect cellular proteins from caspase action. Furthermore, this anti-apoptotic function of CK2 may contribute to its ability to participate in transformation and tumorigenesis.
Water structure in supercritical mixtures of water and rare gases
Authors: Botti A. , Bruni F. , Isopo A. , Modesti G. , Oliva C. , Ricci M. A. , Senesi R. , Soper A. K.
Published: December 2002

The microscopic structure of supercritical solutions of rare gases in water has been determined by neutron diffraction experiments using hydrogen isotope contrast on the water hydrogen atoms, at a concentration of 1 solute atom to ∼40 water molecules. It is found that even at this low concentration and high temperature, rare gases do affect the structure of water, giving a compression of the water–water spatial density functions, which is stronger for the less polarizable solutes. All the mixtures studied exhibit a water deficit around the solute. The sign of the derivative of the critical temperature of the mixture with respect to the critical pressure in the vicinity of the pure solute critical point seems to be correlated with the capability of the solute in penetrating inside the first shell of neighboring water molecules.
Evidence of local and global scaling regimes in thin films deposited by sputtering: An atomic force microscopy and electrochemical study
Authors: Souza Cruz Tersio G. , Kleinke M. U. , Gorenstein A.
Published: December 2002

The surface morphology of NiOx thin films deposited by rf sputtering was studied by atomic force microscopy and by cyclic voltammetry. Linear relationships were observed in log–log plots of the interface width versus window length and in log–log plots of the peak current versus scan rate. Two different slopes were observed, by both techniques, indicating that distinct growth dynamics present in the system can be measured in different ways. Moreover, the calculated fractal dimensions are in excellent agreement: the local scaling regime corresponds to high scan rates and the global scaling regime corresponds to low scan rates, in accordance with the expected behavior for diffusion fronts.
Regulatory CD4+CD25+ T Cells Restrict Memory CD8+ T Cell Responses
Authors: Kursar Mischo , Bonhagen Kerstin , Fensterle Joachim , Köhler Anne , Hurwitz Robert , Kamradt Thomas , Kaufmann Stefan H.E. , Mittrücker Hans-Willi
Published: December 2002

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.
Opsonization of Apoptotic Cells by Autologous iC3b Facilitates Clearance by Immature Dendritic Cells, Down-regulates DR and CD86, and Up-regulates CC Chemokine Receptor 7
Authors: Verbovetski Inna , Bychkov Hila , Trahtemberg Uriel , Shapira Itzhak , Hareuveni Mara , Ben-Tal Ofira , Kutikov Ina , Gill Oranit , Mevorach Dror
Published: December 2002

Immature dendritic cells (iDCs) do not mature after uptake of apoptotic cells and may play a role in the induction of peripheral tolerance to self antigens derived from apoptotic material. The integrins, αvβ3, αvβ5, and the scavenger receptor, CD36, have been shown to mediate uptake of apoptotic cells by iDCs. However, it is not known whether the complement system, also takes part in this process. In this study we investigated the ability of iDCs to bind to apoptotic cells opsonized by iC3b. Monocyte-derived dendritic cells were offered apoptotic Jurkat cells opsonized by autologous iC3b and labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate. A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios. Despite increased efficiency of uptake, interaction between iC3b-opsonized apoptotic cells and iDCs down-regulated the expression of major histocompatibility complex class II, CD86, CC chemokine receptor (CCR)2, CCR5, and β2-integrins (P < 0.001), and up-regulated expression of CCR7 (P < 0.001). In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited. We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.
Genetic Dissection of the Cellular Pathways and Signaling Mechanisms in Modeled Tumor Necrosis Factor–induced Crohn's-like Inflammatory Bowel Disease
Authors: Kontoyiannis Dimitris , Boulougouris George , Manoloukos Menelaos , Armaka Maria , Apostolaki Maria , Pizarro Theresa , Kotlyarov Alexey , Forster Irmgard , Flavell Richard , Gaestel Matthias , Tsichlis Philip , Cominelli Fabio , Kollias George
Published: December 2002

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (TnfΔAREmouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon γ and requires the function of CD8+ T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell–derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor–deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow–derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase–deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.
Chlamydia pneumoniae Infection of the Central Nervous System Worsens Experimental Allergic Encephalitis
Authors: Du Caigan , Yao Song-Yi , Ljunggren-Rose Åsa , Sriram Subramaniam
Published: December 2002

Experimental allergic encephalitis (EAE) is considered by many to be a model for human multiple sclerosis. Intraperitoneal inoculation of mice with Chlamydia pneumoniae, after immunization with neural antigens, increased the severity of EAE. Accentuation of EAE required live infectious C. pneumoniae, and the severity of the disease was attenuated with antiinfective therapy. After immunization with neural antigens, systemic infection with C. pneumoniae led to the dissemination of the organism into the central nervous system (CNS) in mice with accentuated EAE. Inoculation with Chlamydia trachomatis did not worsen EAE and infectious organisms were not seen in the CNS. These observations suggest that dissemination of C. pneumoniae results in localized infection in CNS tissues in animals with EAE. We propose that infection of the CNS by C. pneumoniae can amplify the autoreactive pool of lymphocytes and regulate the expression of an autoimmune disease.
Sustained Activation of Lyn Tyrosine Kinase In Vivo Leads to Autoimmunity
Authors: Hibbs Margaret L. , Harder Kenneth W. , Armes Jane , Kountouri Nicole , Quilici Cathy , Casagranda Franca , Dunn Ashley R. , Tarlinton David M.
Published: December 2002

Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lynup/up mice). Lynup/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lynup/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cγ2 in resting Lynup/up B cells. Similarly, Lynup/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lynup/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.
Acetylation Suppresses the Proapoptotic Activity of GD3 Ganglioside
Authors: Malisan Florence , Franchi Luigi , Tomassini Barbara , Ventura Natascia , Condò Ivano , Rippo Maria Rita , Rufini Alessandra , Liberati Laura , Nachtigall Claudia , Kniep Bernhard , Testi Roberto
Published: December 2002

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.
Lipopolysaccharide-enhanced, Toll-like Receptor 4–dependent T Helper Cell Type 2 Responses to Inhaled Antigen
Authors: Eisenbarth Stephanie C. , Piggott Damani A. , Huleatt James W. , Visintin Irene , Herrick Christina A. , Bottomly Kim
Published: December 2002

Allergic asthma is an inflammatory lung disease initiated and directed by T helper cells type 2 (Th2). The mechanism involved in generation of Th2 responses to inert inhaled antigens, however, is unknown. Epidemiological evidence suggests that exposure to lipopolysaccharide (LPS) or other microbial products can influence the development and severity of asthma. However, the mechanism by which LPS influences asthma pathogenesis remains undefined. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 (Th1) responses, it is unclear if TLRs are needed for Th2 priming. Here, we report that low level inhaled LPS signaling through TLR4 is necessary to induce Th2 responses to inhaled antigens in a mouse model of allergic sensitization. The mechanism by which LPS signaling results in Th2 sensitization involves the activation of antigen-containing dendritic cells. In contrast to low levels, inhalation of high levels of LPS with antigen results in Th1 responses. These studies suggest that the level of LPS exposure can determine the type of inflammatory response generated and provide a potential mechanistic explanation of epidemiological data on endotoxin exposure and asthma prevalence.
Non–T Cell Activation Linker (NTAL)
Authors: Brdička Tomáš , Imrich Martin , Angelisová Pavla , Brdičková Naděžda , Horváth Ondrej , Špička Jiří , Hilgert Ivan , Lusková Petra , Dráber Petr , Novák Petr , Engels Niklas , Wienands Jürgen , Simeoni Luca , Österreicher Jan , Aguado Enrique , Malissen Marie , Schraven Burkhart , Hořejší Václav
Published: December 2002

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non–T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcγ- and Fcε-receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non–T cells.
CD1-mediated γ/δ T Cell Maturation of Dendritic Cells
Authors: Leslie David S. , Vincent Michael S. , Spada Franca M. , Das Hiranmoy , Sugita Masahiko , Morita Craig T. , Brenner Michael B.
Published: December 2002

Immature myeloid dendritic cells (DCs) express only low levels of major histocompatibility complex (MHC) class II but express high levels of CD1 a, b, and c antigen-presenting molecules at the cell surface. As Vδ1+ γ/δ T cells are the main tissue subset of γ/δ T cells and they are known to recognize CD1c in the absence of specific foreign antigen recognition, we examined the possible interaction of these T cells with immature DCs. We show that CD1-restricted γ/δ T cells can mediate the maturation of DCs. DC maturation required cell–cell contact and could be blocked by antibodies against CD1c. The maturation process was partially mediated by tumor necrosis factor α. Importantly, immature DCs matured in the presence of lipopolysaccharide and CD1-restricted γ/δ T cells produced bioactive interleukin-12p70. In addition, these DCs were able to efficiently present peptide antigens to naive CD4+ T cells. CD1-restricted γ/δ T cell recognition of immature DCs provides the human immune system with the capacity to rapidly generate a pool of mature DCs early during microbial invasion. This may be an important source of critical host signals for T helper type 1 polarization of antigen-specific naive T cells and the subsequent adaptive immune response.
The PAAD/PYRIN-Family Protein ASC Is a Dual Regulator of a Conserved Step in Nuclear Factor κB Activation Pathways
Authors: Stehlik Christian , Fiorentino Loredana , Dorfleutner Andrea , Bruey Jean-Marie , Ariza Eugenia M. , Sagara Junji , Reed John C.
Published: December 2002

Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-κB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-κB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-κB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-κB activity, ASC has an inhibitory influence on NF-κB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)α, interleukin 1β, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IκB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IκBα. Our findings suggest that ASC modulates diverse NF-κB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.
Anti–DNA B Cells in MRL/lpr Mice Show Altered Differentiation and Editing Pattern
Authors: Li Yijin , Li Hui , Ni Dongyao , Weigert Martin
Published: December 2002

We have studied the regulation of anti–DNA B cells in transgenic mice with a heavy chain transgene (3H9H/56R). This transgene codes for a heavy chain that forms anti–double-stranded DNA (dsDNA) antibody when paired with most members of the endogenous Vκ repertoire, but certain L chains, referred to as Vκ editors, do not sustain dsDNA binding in combination with 3H9H/56R. In the nonautoimmune 3H9H/56R BALB/c, most B cells generated do not bind DNA because the transgene itself is edited or is associated with a Vκ editor. A minor population of B cells (30%) bind dsDNA and express the λ1 light chain (known to sustain 3H9H/56R DNA binding). These 3H9/56R/λ1 B cells coexpress a κ editor, and we propose that the down-regulation of the anti-DNA BCR caused by the dual L chain expression may prevent activation of this κ/λ population. These κ/λ B cells are sequestered in the marginal zone. Here, we studied the influence of autoimmunity on expression and regulation of 3H9H/56R. In 3H9H/56R MRL/lpr mice, the expression of anti-dsDNA is vastly accelerated. Anti–dsDNA B cells use noneditor κs but, in addition, most anti–dsDNA B cells have edited the heavy chain transgene. λ1 B cells (without the coexpression of a κ editor) are found and the κ/λ1 MZ population is absent. Our results suggest that improper editing and failure to sequester autoreactive B cells may contribute to the breakdown of tolerance in MRL/lpr mice.