bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site
Authors: WANG Wen-Yi , LIAW Shwu-Huey , LIAO Ta-Hsiu
Published: August 2015

Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3ʹ and 5ʹ RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His211 in this conserved motif was shown to be very important in catalysis by site-specific modification with 14C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg2+ and Ca2+. The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.
Alternative splicing for the α1 subunit of soluble guanylate cyclase
Authors: RITTER Detlef , TAYLOR James F. , HOFFMANN Joseph W. , CARNAGHI Lynn , GIDDINGS Stephen J. , ZAKERI Hamideh , KWOK Pui-Yan
Published: August 2015

Soluble guanylate cyclase (sGC), the receptor for nitric oxide, is a heterodimer consisting of α and β subunits. We investigated the mRNA species for the α1 subunit in human brain, heart, artery and immortalized B-lymphocytes. Three mRNA species were identified in these tissues. The major mRNA species contained the full expression sequence of the α1 subunit. Two other types of mRNA were detected in which 5ʹ sequences were deleted by splicing (506-590 and 412-590). Each of these deletions included the predicted translation start site, indicating that translation of these two alternatively spliced RNA species does not result in the production of full-length α1 subunits. The relative amounts of the two mRNA species with deletions of the translation start site differed significantly between cell lines of immortalized B-lymphocytes from different individuals. sGC enzymic activity was significantly decreased in cellular extracts from cell lines with high proportions of mRNA species containing the deletion 506-590 when compared with extracts from cell lines that contained mostly mRNA without this deletion.
Molecular mechanisms of contraction-regulated cardiac glucose transport
Authors: TILL Martin , OUWENS D. Margriet , KESSLER Alexandra , ECKEL Jürgen
Published: August 2015

Insulin and contraction are the most important regulators of glucose utilization in cardiac muscle. In contrast with insulin, the intracellular signalling elements of contraction have remained unexplored. In the present studies, adult rat ventricular cardiomyocytes were electrically stimulated to perform rhythmic contractions to permit the determination of potential sites of convergence of contraction and insulin signalling to glucose transport. The participation of phosphoinositide 3-kinase (PI-3K) in Ca2+- and contraction-stimulated 3-O-methylglucose transport was suggested by the great sensitivity of this process towards the PI-3K inhibitors wortmannin and LY294002 and by the presence of PI-3K activity in anti-phosphotyrosine immunoprecipitates from contracted cells. Initial signalling events of insulin action, including receptor kinase activation, the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 and the recruitment of PI-3K to IRS-1 and IRS-2, were found not to be involved in contraction-mediated signalling. However, immunoprecipitation of p85α revealed a markedly enhanced tyrosine phosphorylation of an unknown co-precipitated 200 kDa protein in response to both stimuli. It is concluded that contraction-regulated cardiac glucose transport involves the activation of PI-3K in response to upstream signalling pathways different from that of insulin.
Identification of Nω-carboxymethylarginine as a novel acid-labile advanced glycation end product in collagen
Authors: IIJIMA Katsumasa , MURATA Michio , TAKAHARA Hidenari , IRIE Shinkichi , FUJIMOTO Daisaburou
Published: August 2015

Collagen undergoes continuous non-enzymatic glycation during its long life period. The products resulting from the glycation reaction, so-called advanced glycation end products (AGEs), were regarded as potential pathogens of various diseases such as diabetic complications. Although several AGEs were identified from acid hydrolysates of glycated collagen, the major AGE(s) responsible for the diseases have not yet been fully characterized. Moreover, acid-labile constituents were decomposed during acid hydrolysis. To investigate these AGEs, we used the enzymatic hydrolysis method [Bensusan, Dixit and McKnight (1971) Biochim. Biophys. Acta 251, 100-108]. As a result, an acid-labile unknown compound was discovered from the digested glycated collagen. We identified this compound as NΩ-carboxymethylarginine (CMA) by matrix-assisted laster-desorption ionization-MS and NMR. CMA gradually increased in collagen during incubation with glucose and the yield reached about 8 mol/mol of collagen, which is 100 times higher than that of pentosidine. This result suggests that CMA is a major AGE in collagen.
Phosphorylation of Oct-2 at sites located in the POU domain induces differential down-regulation of Oct-2 DNA-binding ability
Authors: PEVZNER Veniamin , KRAFT Regine , KOSTKA Susanne , LIPP Martin
Published: August 2015

We compared the effects of phosphorylation of Oct-2 protein on its binding to the consensus octamer sequence (ATGCAAAT) and two non-canonical sequences present in human (AAGCAAAT) and murine (AAACAAAT) promoters of the BLR1 (Burkitts' lymphoma receptor 1) gene encoding chemokine receptor CXCR5 (CXC-chemokine receptor 5). The latter cis-acting elements represent low-affinity recognition sequences for the octamer transcription factors. Okadaic acid was found to induce hyperphosphorylation of Oct-2 specifically in cells of lymphoid lineage. Potentially phosphorylated amino acid residues localized to the POU-specific domain of Oct-2. Whereas binding of Oct-2 to the octamer site from the human BLR1 promoter or to the consensus octamer sequence was unaffected by phosphorylation of this factor, a strong reduction of Oct-2 binding to the octamer site from the murine BLR1 promoter was observed. This finding correlates well with the down-regulation of expression of the BLR1 gene in murine splenic cells but not in lymphoid cells of human origin treated with okadaic acid. These data support the hypothesis that phosphorylation of Oct-2 may be a mechanism by which activities of the promoters containing non-canonical octamer sequences are differentially regulated in response to extracellular stimuli.
Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases
Authors: ROSSIGNOL Rodrigue , LETELLIER Thierry , MALGAT Monique , ROCHER Christophe , MAZAT Jean-Pierre
Published: August 2015

Metabolic control analysis has often been used for quantitative studies of the regulation of mitochondrial oxidative phosphorylations (OXPHOS). The main contribution of this work has been to show that the control of mitochondrial metabolic fluxes can be shared among several steps of the oxidative phosphorylation process, and that this distribution can vary according to the steady state and the tissue. However, these studies do not show whether this observed variation in the OXPHOS control is due to the experimental conditions or to the nature of the mitochondria. To find out if there actually exists a tissue variation in the distribution of OXPHOS control coefficients, we determined the control coefficients of seven OXPHOS complexes on the oxygen-consumption flux in rat mitochondria isolated from five different tissues under identical experimental conditions. Thus in this work, only the nature of the mitochondria can be responsible for any variation detected in the control coefficient values between different tissues. The analysis of control coefficient distribution shows two tissue groups: (i) the muscle and the heart, controlled essentially at the level of the respiratory chain; and (ii) the liver, the kidney and the brain, controlled mainly at the phosphorylation level by ATP synthase and the phosphate carrier. We propose that this variation in control coefficient according to the tissue origin of the mitochondria can explain part of the tissue specificity observed in mitochondrial cytopathies.
Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components
Authors: SCHURINGA Jan-Jacob , JONK Luigi J. C. , DOKTER Wim H. A. , VELLENGA Edo , KRUIJER Wiebe
Published: August 2015

In the present study, signal transducer and activator of transcription 3 (STAT3) Ser727 phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-terminal kinase (JNK)-1 and p38 (‘reactivating kinase’) in response to interleukin (IL)-6 stimulation. Although IL-6 can activate ERK-1 in HepG2 cells, STAT3 transactivation and Ser727 phosphorylation were not reduced by using the MAP kinase/ERK kinase (MEK) inhibitor PD98059 or by overexpression of dominant-negative Raf. IL-6 did not activate JNK-1 in HepG2 cells and STAT3 was a poor substrate for JNK-1 activated by anisomycin, excluding a role for JNK1 in IL-6-induced STAT3 activation. However, SEK-1/MKK-4 [where SEK-1 stands for stress-activated protein kinase (SAPK)/ERK kinase 1, and MKK-4 stands for MAP kinase kinase 4] was activated in response to IL-6 and overexpression of dominant-negative SEK-1/MKK-4(A-L) reduced both IL-6-induced STAT3 Ser727 phosphorylation as well as STAT3 transactivation. Subsequently, the SEK-1/MKK-4 upstream components Vav, Rac-1 and MEKK were identified as components of a signal transduction cascade that leads to STAT3 transactivation in response to IL-6 stimulation. Furthermore, inhibition of p38 kinase activity with the inhibitor SB203580 did not block STAT3 Ser727 phosphorylation but rather increased both basal as well as IL-6-induced STAT3 transactivation, indicating that p38 may act as a negative regulator of IL-6-induced STAT3 transactivation through a presently unknown mechanism. In conclusion, these data indicate that IL-6-induced STAT3 transactivation and Ser727 phosphorylation is independent of ERK-1 or JNK-1 activity, but involves a gp130 receptor-signalling cascade that includes Vav, Rac-1, MEKK and SEK-1/MKK-4 as signal transduction components.
Regulation of the gene promoter for extracellular signal-regulated protein kinase 2 by transcription factors NF-Y and Sp3
Authors: SUGIURA Naoaki , TAKISHIMA Kunio
Published: August 2015

We have previously shown that the maximal promoter activity of the gene for extracellular signal-regulated protein kinase 2 (ERK2; also known as p42 mitogen-activated protein kinase) resides in the 371 bp 5ʹ-flanking sequence. In the present study we defined roles for a CCAAT box and two adjacent GC boxes in the activity of this promoter. Deletion analysis and DNase I footprinting of this 371 bp region indicated that the CCAAT box at -64 and GC boxes at -86 and -39 are crucial for promoter activity. Electrophoretic mobility-shift assays showed that transcription factor NF-Y/CBF binds to the CCAAT box. Sp1 and Sp3, members of the Sp family of transcription factors, bind to the GC boxes of the ERK2 promoter. The binding of Sp3 was predominant over that of Sp1. Disruption by mutation of any of the CCAAT box and GC boxes similarly decreased promoter activity. These three cis elements exhibited a moderate synergy in promoter function. The transactivating role of NF-Y was corroborated by the finding that a dominant-negative form of NF-YA diminished the promoter activity. These results provide clues for refining our understanding of not only the regulation of expression of the gene for ERK2 but also mechanisms by which NF-Y and Sp1/Sp3 regulate transcription.
Electrostatic interactions affecting the active site of class Sigma glutathione S-transferase
Authors: STEVENS Julie M. , ARMSTRONG Richard N. , DIRR Heini W.
Published: August 2015

We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the α-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.
Cytokine-inducible enhancer with promoter activity in both the rat and human manganese-superoxide dismutase genes
Authors: ROGERS Richard J. , CHESROWN Sarah E. , KUO Shiuhyang , MONNIER Joan M. , NICK Harry S.
Published: August 2015

Diverse pro-inflammatory mediators regulate transcription of the gene (MnSOD) encoding the mitochondrial anti-oxidant protein manganese-superoxide dismutase. Understanding the regulation of this gene is crucial to comprehending its role in cytoprotection. In transfected lung epithelial cells, a human-growth-hormone reporter gene system was utilized to identify a potential enhancer in the MnSOD genomic fragment previously shown to contain multiple DNase-I-hypersensitive sites. Northern analysis demonstrated a 10-20-fold increase in response to pro-inflammatory mediators. Inclusion of the MnSOD genomic fragment in reporter constructs was necessary to mimic these stimulus-dependent endogenous levels. The inducible enhancer element was localized to a 260 bp fragment in intron 2, coinciding with a previously defined DNase-I-hypersensitive site. This element functions in an orientation- and position-independent manner as well as with the heterologous thymidine kinase promoter. In addition, we have demonstrated that a homologous sequence within the human MnSOD gene exhibits identical enhancer activity. A novel characteristic of the rat and human enhancer elements involves the ability to promote cytokine-inducible transcription in the absence of a classical promoter.
Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites
Authors: MARCHESINI Norma , LUO Shuhong , RODRIGUES Claudia O. , MORENO Silvia N. J. , DOCAMPO Roberto
Published: August 2015

Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1, nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2+, and (2) the effect of ionomycin, which cannot take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A1, nigericin, monensin, or NH4Cl. Inorganic PPi promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PPi collapsed by addition of the K+/H+ exchanger, nigericin, and eliminated by the PPi analogue, aminomethylenediphosphonate (AMDP). Both PPi hydrolysis and proton transport were dependent upon K+, and Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H+-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca2+]i in the nominal absence of extracellular Ca2+. Ionomycin was more effective in releasing Ca2+ from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H+-PPase and V-H+-ATPase in these organelles.
PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum
Authors: BRACCHI-RICARD Valerie , BARIK Sailen , DELVECCHIO Cherie , DOERIG Christian , CHAKRABARTI Ratna , CHAKRABARTI Debopam
Published: August 2015

We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T173PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2+ ions as a cofactor. The Lys38 → Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.
Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression
Authors: FRANK Stefan , KÄMPFER Heiko , WETZLER Christian , STALLMEYER Birgit , PFEILSCHIFTER Josef
Published: August 2015

We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1β- and tumour-necrosis-factor-α-induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-NG-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair.
Specific role of the extracellular signal-regulated kinase pathway in angiotensin II-induced cardiac hypertrophy in vitro
Authors: AOKI Hiroki , RICHMOND Mary , IZUMO Seigo , SADOSHIMA Junichi
Published: August 2015

Although MAP (mitogen-activated protein) kinases are implicated in cell proliferation and differentiation in many cell types, the role of MAP kinases in cardiac hypertrophy remains unclear. We examined the role of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAP kinase in angiotensin II (Ang II)-induced hypertrophy compared with phenylephrine-induced hypertrophy in neonatal rat cardiac myocytes. Both Ang II and phenylephrine activated ERKs to a similar extent, whereas phenylephrine caused stronger and more sustained activation of JNK and p38 than Ang II. PD98059, a specific inhibitor of MAPK/ERK kinase (MEK),inhibited Ang II-induced, but not phenylephrine-induced, expression of atrial natriuretic factor (ANF) at both the mRNA and polypeptide levels. SB203580, a specific inhibitor of p38 and some JNK isoforms, did not show significant effects on ANF expression induced by Ang II or phenylephrine. Although PD98059 and dominant-negative MEK1 blocked Ang II-induced activation of the ANF promoter, SB203580 or dominant-negative MEK kinase 1 (MEKK1) showed no effect. Phenylephrine-induced ANF promoter activation was significantly inhibited by SB203580 and dominant-negative MEKK1, but not by PD98059 or dominant-negative MEK1. Dominant-negative Ras inhibited both ERK activation and ANF up-regulation by Ang II, whereas constitutively active forms of Ras and MEK were sufficient to activate the ANF promoter. Dominant-negative Ras also partly inhibited the phenylephrine-induced activation of ANF promoter. PD98059 did not affect other markers of Ang II-induced hypertrophy, such as skeletal α-actin and c-fos expression, increases in the rate of protein synthesis or rapid sarcomeric actin organization. These results suggest that Ang II uses ERK for ANF expression, whereas phenylephrine uses other pathways. The Ras/ERK pathway selectively mediates ANF expression in various phenotypes observed in Ang II-induced hypertrophy. The ERK pathway mediates an agonist-specific and phenotype-specific response in cardiac hypertrophy.
Properties of a polyamine transporter regulated by antizyme
Authors: SAKATA Kaori , KASHIWAGI Keiko , IGARASHI Kazuei
Published: August 2015

The regulation of polyamine transport by antizyme, a protein that is involved in the rapid degradation of ornithine decarboxylase (ODC), was studied in FM3A mouse cells overproducing ODC. Both artificial (Z1) and natural antizymes not only inhibited polyamine uptake but also stimulated polyamine excretion. The properties of the polyamine transporter regulated by antizyme were characterized. The uptake of radiolabelled polyamines was inhibited by excess acetylpolyamines and a protonophore, CCCP (carbonyl cyanide m-chlorophenylhydrazone), whereas the excretion of radiolabelled polyamines was stimulated by unlabelled polyamines, acetylpolyamines and CCCP in the medium. Furthermore, it is shown that polyamines and acetylpolyamines are excreted from cells. On the basis of the results, it is discussed how antizyme regulates polyamine transport negatively.
Among translational effectors, p70S6k is uniquely sensitive to inhibition by glucocorticoids
Authors: SHAH O. Jameel , KIMBALL Scot R. , JEFFERSON Leonard S.
Published: August 2015

Fundamental cellular processes such as cell differentiation and growth, apoptosis and cellular metabolism are regulated differentially by glucocorticoid hormones in a cell-context-related fashion. However, these basic processes are not governed by isolated signals but are influenced by the integration of both synergistic and antagonistic extracellular and intracellular stimuli. Because glucocorticoids and insulin-like growth factor I (IGF-I) reciprocally modulate growth-regulated processes such as translation initiation, especially in skeletal muscle, a study was undertaken to address the nature of this counter-regulation. Quiescent L6 skeletal myoblasts pretreated for 4 h with the synthetic glucocorticoid dexamethasone exhibited a marked attenuation of IGF-I-induced activation of the ribosomal protein S6 kinase (p70S6k). The adverse effects of glucocorticoids on the activity of the endogenous enzyme were due to differential dephosphorylation at discrete residues, suggesting that, physiologically, some but not all phosphorylation sites are subject to mitogenic regulation. Furthermore, the translational repressor eIF4E-binding protein 1 (4E-BP1), which in many circumstances is co-ordinately regulated with p70S6k, was dephosphorylated in response to glucocorticoids; however, hyperphosphorylation of the protein after stimulation with IGF-I was refractory to inhibition by glucocorticoids, as was its dissociation from its binding partner, eIF4E. Although both basal and IGF-I-stimulated rates of protein synthesis were modestly affected by glucocorticoids, the synthesis of EF1A, whose mRNA precursor is a prototype for the terminal oligopyrimidine (‘TOP’) transcript family and whose expression is controlled by the activity of p70S6k, was markedly affected. Therefore in this cell system it seems that, despite the mutual control of p70S6k and 4E-BP1 that is often observed, p70S6k is more sensitive to down-regulation by glucocorticoids under growth-promoting conditions than is 4E-BP1.
Glucocorticoid regulation of branched-chain α-ketoacid dehydrogenase E2 subunit gene expression
Authors: COSTEAS Paul A. , CHINSKY Jeffrey M.
Published: August 2015

Regulation of the mammalian branched-chain α-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, α-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1α subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5ʹ proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5ʹ upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors.
Thrombospondin-1 binds to polyhistidine with high affinity and specificity
Authors: VANGURI Vijay K. , WANG Shuxia , GODYNA Svetlana , RANGANATHAN Sripriya , LIAU Gene
Published: August 2015

Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a 125I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of 125I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a Ki of 50 nM, suggesting that the heparin-binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study.
Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine–DNA alkyltransferase insensitive to O6-benzylguanine
Authors: XU-WELLIVER Meng , PEGG Anthony E.
Published: August 2015

The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O6-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials. Point mutations in a 24 amino acid sequence likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserved residues Lys165, Leu168 and Leu169. The two positions at which changes led to the largest increase in resistance to BG were Gly156 and Lys165. Eleven mutants at Gly156 were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lys165 found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Ser159 position (S159I and S159V) were > 80-fold more resistant than wild-type AGT. Eleven active mutants at Leu169 were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cys150, with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation. Six BG-resistant mutants at Asn157 were found with increases of 4-13-fold. These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents.
Order and maximum incorporation of N-acetyl-d-galactosamine into threonine residues of MUC2 core peptide with microsome fraction of human-colon-carcinoma LS174T cells
Authors: IIDA Shin-ichiro , TAKEUCHI Hideyuki , KATO Kentaro , YAMAMOTO Kazuo , IRIMURA Tatsuro
Published: August 2015

Mucin 2 (MUC2) is the major intestinal mucin. O-glycans are attached to MUC2 in a potentially diverse arrangement, which is crucial for their interaction with endogeneous and exogeneous lectins. In the present report, five oligopeptides [PTTTPITTTT(K), ITTTTTVTPT(K), TVTPTPTPTG(K), PTPTGTQTPT(K) and TQTPTTTPIT(K)] corresponding to portions of the MUC2 tandem repeat domain were synthesized, and incubated with UDP-N-acetyl-D-galactosamine (UDP-GalNAc) and detergent-soluble microsomes, prepared from the human colon carcinoma cell line LS174T. The products were fractionated by reverse-phase HPLC and characterized by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Oligopeptides with GalNAc residues derived from PTTTPITTTT(K), containing consecutive threonine residues, were found to be glycosylated with 1-7 GalNAc residues per single peptide. The sequences of all glycopeptides were determined. The results indicated that the predominant sites of the first through to the sixth GalNAc incorporation were Thr3, Thr6, Thr5, Thr2, Thr4 and Thr1, respectively. An exception was the presence of a glycopeptide with three GalNAc residues at Thr1, Thr4 and Thr5. Oligopeptides containing alternating threonine residues [TVTPTPTPTG(K) and PTPTGTQTPT(K)] were not fully glycosylated under the same conditions or even after prolonged incubations. Thus, the preferential order and maximum number of GalNAc incorporation into threonine residues of MUC2 core peptides depends on the peptide sequence, when the microsome fraction of LS174T cells is used as a source of N-acetyl-D-galactosaminyltransferases.
Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of β-d-glucose
Authors: WITT Susanne , WOHLFAHRT Gerd , SCHOMBURG Dietmar , HECHT Hans-Jürgen , KALISZ Henryk M.
Published: August 2015

The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated. Kinetic studies on the oxidation of β-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of β-D-glucose, to be absolutely essential for the efficient binding of β-D-glucose. The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of β-D-glucose, exhibits an 80-fold higher apparent Km (513 mM) but a Vmax only 70% lower (280 units/mg) than the wild type. The complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of β-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent Km for glucose (to 733 mM) and a decrease in the Vmax to 1/30 (33 units/mg). None of the other substitutions, with the exception of variant F418A, affected the apparent Km more than 6-fold. In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90. Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely, inactive. Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of β-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation.
The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk
Authors: KURAISHI Takeshi , SUN Youping , AOKI Fugaku , IMAKAWA Kazuhiko , SAKAI Senkiti
Published: August 2015

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of β- and γ-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P < 0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P < 0.05). The β- and γ-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.
Large-scale synthesis and functional elements for the antimicrobial activity of defensins
Authors: RAJ Periathamby Antony , ANTONYRAJ Kavitha J. , KARUNAKARAN Thonthi
Published: August 2015

Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic β-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.
Differential adrenergic regulation of the gene expression of the β-adrenoceptor subtypes β1, β2 and β3 in brown adipocytes
Authors: BENGTSSON Tore , CANNON Barbara , NEDERGAARD Jan
Published: August 2015

In brown adipocytes, fundamental cellular processes (cell proliferation, differentiation and apoptosis) are regulated by adrenergic stimulation, notably through β-adrenergic receptors. The presence of all three β-receptor subtypes has been demonstrated in brown adipose tissue. Due to the significance of the action of these receptors and indications that the subtypes govern different processes, the adrenergic regulation of the expression of the β1-, β2- and β3-adrenoceptor genes was examined in murine brown-fat primary cell cultures. Moderate levels of β1-receptor mRNA, absence of β2-receptor mRNA and high levels of β3-receptor mRNA were observed in mature brown adipocytes (day 6 in culture). Noradrenaline (norepinephrine) addition led to diametrically opposite effects on β1- (markedly enhanced expression) and β3-gene expression (full cessation of expression, as previously shown). β2-Gene expression was induced by noradrenaline, but only transiently (< 1 h). The apparent affinities (EC50) of noradrenaline were clearly different (7 nM for the β1-gene and≤ 1 nM for the β3-gene), as were the mediation pathways (solely via β3-receptors and cAMP for the β1-gene and via β3-receptors and cAMP, as well as via α1-receptors and protein kinase C, for the β3-gene). The half-lives of the corresponding mRNA species were very short but different (17 min for β1-mRNA and 27 min for β3-mRNA), and these degradation rates were not affected by noradrenaline, implying that the mRNA levels were controlled by transcription. Inhibition of protein synthesis also led to diametrically opposite effects on β1- and β3-gene expression, but - notably - these effects were congruent with the noradrenaline effects, implying that a common factor regulating β1-gene expression negatively and β3-gene expression positively could be envisaged. In conclusion, very divergent effects of adrenergic stimulation on the expression of the different β-receptor genes were found within one cell type, and no unifying concept of adrenergic control of β-receptor gene expression can be formulated, either concerning different cell types, or concerning the different β-receptor subtype genes.
Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown
Authors: POWERS Elaine A. , THOMPSON David P. , GARNER-HAMRICK Peggy A. , HE Wanxia , YEM Anthony W. , BANNOW Carol A. , STAPLES Douglas J. , WASZAK Gregory A. , SMITH Clark W. , DEIBEL Martin R. , FISHER Chris
Published: August 2015

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.
Binding of α2-macroglobulin and limulin: regulation of the plasma haemolytic system of the American horseshoe crab, Limulus
Authors: SWARNAKAR Snehasikta , ASOKAN Rengasamy , QUIGLEY James P. , ARMSTRONG Peter B.
Published: August 2015

The mediator of haemolysis in the plasma of the horseshoe crab, Limulus polyphemus, is limulin, a sialic acid-binding lectin. The haemolytic activity of limulin is inhibited by thiol ester-reacted forms of Limulus α2-macroglobulin, the third-most abundant protein of the plasma. Limulus α2-macroglobulin that has experienced cleavage of its internal thiol ester bond, consequent to reaction with proteases, or with the small primary amine, methylamine, reduces the haemolytic activity of limulin when present at molar excesses that approximate the relative concentrations of these two proteins in the plasma. Native, unreacted Limulus α2-macroglobulin has no effect on the haemolytic activity of limulin. Limulin binds thiol ester-reacted forms of Limulus α2-macroglobulin both in a solid-phase assay and in solution with an avidity 10-25 times higher than native, unreacted Limulus α2-macroglobulin. Protease-reacted α2-macroglobulin functions as a marker for the presence of foreign proteases in the blood of Limulus, and thus of pathogenic organisms that release proteases as facilitators of invasion and pathogenicity. Modulation of the haemolytic system represents a novel function for α2-macroglobulin.
Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (eIF2), absent from eIF5, mediate interaction with eIF2γ
Authors: THOMPSON Glória M. , PACHECO Eliza , MELO Eduardo O. , CASTILHO Beatriz A.
Published: August 2015

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5ʹ end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2β homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues. eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.