bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens
Authors: Westman Erin L. , Mcnally David J. , Rejzek Martin , Miller Wayne L. , Kannathasan Vellupillai Sri , Preston Andrew , Maskell Duncan J. , Field Robert A. , Brisson Jean-Robert , Lam Joseph S. 
Published: August 2015

The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6–WbpI and His6–WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6–WbpI or His6–WlbD reactions with UDP-α-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme–substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6–WbpI catalysed the 2-epimerization of UDP-α-D-GlcNAc3NAcA, converting it into UDP-α-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases.
Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?
Authors: Hornemann Thorsten , Wei Yu , von Eckardstein Arnold 
Published: August 2015

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1–3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.
The human NUPR1/P8 gene is transcriptionally activated by transforming growth factor β via the SMAD signalling pathway
Authors: Pommier Roxane M. , Gout Johann , Vincent David F. , Cano Carla E. , Kaniewski Bastien , Martel Sylvie , Rodriguez Jonathan , Fourel Geneviève , Valcourt Ulrich , Marie Julien C. , Iovanna Juan L. , Bartholin Laurent 
Published: August 2015

NUPR1 (nuclear protein 1), also called P8 (molecular mass 8 kDa) or COM1 (candidate of metastasis 1), is involved in the stress response and in cancer progression. In the present study, we investigated whether human NUPR1 expression was regulated by TGFβ (transforming growth factor β), a secreted polypeptide largely involved in tumorigenesis. We demonstrate that the expression of NUPR1 was activated by TGFβ at the transcriptional level. We show that this activation is mediated by the SMAD proteins, which are transcription factors specifically involved in the signalling of TGFβ superfamily members. NUPR1 promoter analysis reveals the presence of a functional TGFβ-response element binding the SMAD proteins located in the genomic DNA region corresponding to the 5′-UTR (5′-untranslated region). Altogether, the molecular results of the present study, which demonstrate the existence of a TGFβ/SMAD/NUPR1 activation cascade, open the way to consider and investigate further a new mechanism enabling TGFβ to promote tumorigenesis by inducing stress resistance.
Identification and distribution of proteins in isolated endosomal fractions of rat liver: involvement in endocytosis, recycling and transcytosis
Authors: POL Albert , ORTEGA David , ENRICH Carlos 
Published: August 2015

1. The polypeptides of the three endosomal fractions isolated from livers of oestradiol-treated rats were analysed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approx. 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated.
2. The ‘early’ endosomes [compartment of uncoupling of receptors and ligands (CURL)] comprised the most complex fraction and contained most of the polypeptides found in the ‘late’ endosomes [multivesicular bodies (MVBs)] and the receptor recycling compartment (RRC). When CURL was analysed by two-dimensional gel electrophoresis after partition with Triton X-114, it showed the largest number of integral membrane polypeptides.
3. Some of the major receptors (polymeric immunoglobulin receptor, transferrin receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, β1-integrin, mannose 6-phosphate receptor, epidermal growth factor receptor and AGp110) and internalized ligands (IgA, IgG, albumin, haptoglobin, transferrin and α2-macroglobulin) were further studied by Western blotting.
4. The distribution of the identified receptors and ligands among the three endosomal fractions was in agreement with their expected functionalities.
5. The polypeptide composition of the bile was also examined and compared with ligands and proteins identified in the different endocytic fractions.
6. Finally, an electron microscopy study confirms the distinctive physical and ultrastructural features of the three isolated endosomal fractions.
Inhibition of cytokine-induced inducible nitric oxide synthase expression by glucagon and cAMP in cultured hepatocytes
Authors: SMITH Fiona S. , CEPPI Enrico D. , TITHERADGE Michael A. 
Published: August 2015

Addition of lipopolysaccharide plus interferon γ, tumour necrosis factor α and interleukin 1β to cultured hepatocytes resulted in the induction of inducible nitric oxide synthase (iNOS) activity as measured by NO3-+NO2- formation, the conversion of L-[U-14C]arginine into citrulline and Western blotting of the iNOS protein. The inclusion of 1 μM glucagon during the induction period significantly decreased the effect of the cytokines on iNOS activity, the major effect being at the level of the total amount of protein, rather than alterations in substrate supply or covalent modification of the existing protein. In contrast, 1 μM insulin was without effect. The effect of glucagon was mediated via cAMP and could be mimicked by the presence of either dibutyryl cAMP or forskolin to activate adenylate cyclase directly. It was rapid in onset and long-lived, a 30 min pre-treatment period protecting the cells from the induction of NO synthesis over the next 21 h in the presence of cytokines. Addition of glucagon at any time point up to 9 h after treatment of the cells with lipopolysaccharide plus the cytokines resulted in a significant inhibition of iNOS activity, glucagon being most potent when added during the first 3 h.
Co-operation of the 5′ and 3′ untranslated regions of ornithine decarboxylase mRNA and inhibitory role of its 3′ untranslated region in regulating the translational efficiency of hybrid RNA species via cellular factor(s)
Authors: LORENZINI Erna C. , SCHEFFLER Immo E. 
Published: August 2015

The 5′ untranslated region (UTR) has an inhibitory role in the translatability of ornithine decarboxylase (ODC) mRNA and of hybrid mRNA species, whereas the ODC 3′ UTR causes a partial release of this inhibition. We designed experiments to explore whether the co-operation between ODC 5′ UTR and 3′ UTR in the translational regulation is due to a direct interaction of those sequences or whether it is mediated by their interaction with cellular factor(s). We stably transfected Chinese hamster ovary (CHO)-K1 cells and transiently transfected COS-1 cells with expression vectors carrying different chimaeric DNAs having the luciferase (LUC) coding sequence as reporter gene, the ODC 5′ UTR or the ODC 3′ UTR, or both, in the appropriate positions. We compared the results obtained by assaying the LUC activities of both transfected cell lines with each chimaeric DNA with those observed by translating the hybrid RNAs in a translation system in vitro. When the ODC 3′ UTR was present, we observed a partial release of the translation inhibition owing to the ODC 5′ UTR only in vivo. The releasing effect was restored in vitro by the addition of cytoplasmic extracts from wild-type CHO-K1 or COS-1 cells, prepared 2 and 8 h after their release from serum starvation. We also observed a partial inhibition of the translatability of the hybrid RNA owing to the presence of the ODC 3′ UTR itself; the translational efficiency could be rescued by cell extract from 8 h serum-stimulated cells. The co-operation between the ODC-UTRs might be mediated by factors expressed by cells during particular phases of the cell cycle. Excess copies of the ODC-UTRs, expressed in trans, could compete in binding limited amounts of such regulatory factors and remove them from interaction with the endogenous ODC mRNA. This phenomenon should be reflected by modifications of the kinetics of ODC and/or LUC activities during serum stimulation. The overexpression of the ODC 3′ UTR determined an increase in both endogenous ODC activity and LUC activity. Moreover, in the transfectants expressing the hybrid RNA species bearing the ODC 3′ UTR the basal ODC activity is higher than that observed in control cells. We suggest that excess copies of the ODC 3′ UTR mis-regulate the endogenous ODC translatability, probably by tying up regulatory molecules expressed by cells in limited amounts and sequestering them from the ODC mRNA species they should interact with.
Direct modulation of G-proteins by polyunsaturated fatty acids: a novel eicosanoid-independent regulatory mechanism in the developing lung
Authors: MUKHOPADHYAY Somnath , RAMMINGER Sarah J. , McLAUGHLIN Mark , GAMBLING Lorraine , OLVER Richard E. , KEMP Paul J. 
Published: August 2015

Basal and fatty-acid-modulated G-protein function was studied in 1–3-day-pre-term, fetal guinea-pig, type II (fATII) pneumocyte apical membrane. Unstimulated (tonic) high-affinity GTPase activity (measured as [γ-32P]GTP hydrolysis rate) was high and 77% pertussis toxin-insensitive. Alteration of this activity was used as a marker of G-protein regulation. Arachidonic acid (AA) showed a dose-dependent (IC50 = 48±8 μM) inhibition of activity at concentrations significantly below critical micellar concentrations; this effect was mimicked by other polyunsaturated fatty acids (IC50 for linoleic acid = 47±2 μM; IC50 for oleic acid= 106±11 μM). Saturated fatty acids showed no effect. The effect of AA on ouabain-insensitive ATPases in the same preparation was significantly lower, suggesting a specificity of the GTPase modulation effect. AA modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5′-lipoxygenase, cyclo-oxygenase and P-450. In order to explore further the mechanism of AA–G-protein interaction, the effect of AA on the time course and equilibrium binding of [35S]GTP[S] to apical membrane was studied. Consistent with our GTPase assay data, AA inhibited binding with an IC50 value of 71±1 μM; stearic acid did not mimic this effect. This is the first report of unsaturated-fatty-acid-specific modulation of lung G-protein function: since AA also up-regulates perinatal lung alveolar Na+ transport, we suggest this lipid/G-protein switch helps maintain pulmonary fluid homoeostasis around birth.
Stat1 associates with c-kit and is activated in response to stem cell factor
Authors: DeBERRY Candy , MOU Sherry , LINNEKIN Diana 
Published: August 2015

Interaction of stem cell factor (SCF), a haematopoietic growth factor, with the receptor tyrosine kinase c-kit leads to autophosphorylation of c-kit as well as tyrosine phosphorylation of various substrates. Little is known about the role of the JAK/STAT pathway in signal transduction via receptor tyrosine kinases, although this pathway has been well characterized in cytokine receptor signal transduction. We recently found that the Janus kinase Jak2 associates with c-kit and that SCF induces rapid and transient phosphorylation of Jak2. Here we present evidence that SCF activates the transcription factor Stat1. Phosphorylated c-kit co-immunoprecipitates with Stat1 within 1 min of SCF stimulation of the human cell line MO7e. Co-precipitation experiments using glutathione S-transferase fusion proteins indicate that association with c-kit is mediated by the Stat1 SH2 domain. Stat1 is rapidly tyrosine-phosphorylated in response to SCF in MO7e cells, the murine cell line FDCP-1 and normal progenitor cells. SCF-induced phosphorylation of Jak2 and Stat1 was also observed in murine 3T3 fibroblasts stably transfected with full-length human c-kit receptor. Furthermore c-kit directly phosphorylates Stat1 fusion proteins in in vitro kinase assays. Electrophoretic mobility-shift assays with nuclear extracts from SCF-stimulated cell lines and normal progenitor cells indicate that activated Stat1 binds the m67 oligonucleotide, a high-affinity SIE promoter sequence. These results demonstrate that Stat1 is activated in response to SCF, and suggest that Stat1 is a component of the SCF signal-transduction pathway.
Co-oxidation of NADH and NADPH by a mammalian 15-lipoxygenase: inhibition of lipoxygenase activity at near-physiological NADH concentrations
Authors: O'DONNELL Valerie B. , KÜHN Hartmut 
Published: August 2015

The purified 15-lipoxygenase from rabbit reticulocytes is capable of oxidizing NADH in the presence of linoleic acid and oxygen. This co-oxidation proceeds at a rate that amounts to approx. 7% of linoleic acid oxygenation rates. Although NADH inhibits the lipoxygenase reaction with linoleic acid as substrate (46% inhibition at 0.2 mM NADH), the reaction specificity of the enzyme was not altered since (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid was identified as the major reaction product. NADH oxidation was inhibited by NAD+ (uncompetitive with respect to linoleate and mixed/competitive with respect to NADH), and NADPH or NMNH could substitute for NADH with slightly different apparent Km values. NADH oxidation was enhanced at lower oxygen tension, but was completely prevented under anaerobic conditions. Computer-assisted modelling of 15-lipoxygenase/NADH interaction and sequence alignments of mammalian lipoxygenases with NADH-dependent enzymes suggested that there is no specific binding of the coenzyme at the putative fatty acid-binding site of lipoxygenases. These results suggest that NAD(P)H might be oxidized by a radical intermediate formed during the dioxygenase cycle of the lipoxygenase reaction but that NADH oxidation might not proceed at the active site of the enzyme. The mechanism and possible biological consequences of 15-lipoxygenase-catalysed NAD(P)H oxidation are discussed.
Mechanism of cAMP-induced Ca2+ influx in Dictyostelium: role of phospholipase A2
Authors: SCHALOSKE Ralph , MALCHOW Dieter 
Published: August 2015

cAMP-induced Ca2+ influx in Dictyostelium follows two pathways: a G-protein-dependent pathway where influx is reduced by 50–70% in Gα2 and Gβ-negative strains and a heterotrimeric G-protein-independent pathway. Using a pharmacological approach, we found that phospholipase A2 (PLA2) is the target of both pathways. The products of PLA2 activity, arachidonic acid (AA) and palmitic acid, induced Ca2+ influx to a similar extent as cAMP. Half-maximal activation occurred at 3 μM AA and saturation at 10 μM AA. The response to AA was quantitatively similar throughout early differentiation and thusindependent of cAMP-receptor concentration. Synergy experiments revealed that cAMP and AA acted through identical pathways. The PLA2-activating peptide, a peptide with sequence similarity to the G-protein β-subunit, activated Ca2+ influx. The G-protein-independent pathway was sensitive to genistein but not to blockers of protein kinase C and other kinases, suggesting that tyrosine kinase may directly or indirectly activate PLA2 in this case.
Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase
Authors: ROUGHAN P. Grattan 
Published: August 2015

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10–20 μM, assuming a stromal volume of 66 μl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 μM. Malonyl-CoA concentrations were also very low (&lt; 0.1–3.0 μM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 μM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA–acyl-carrier-protein transacylase reactions at the concentrations of metabolites measured here are &lt; 0.1–4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.
Metabolism and apoptotic properties of elevated ceramide in HT29rev cells
Authors: VELDMAN Robert J. , KLAPPE Karin , HOEKSTRA Dick , KOK Jan W. 
Published: August 2015

Ceramide (Cer) has been implicated in the regulation of apoptosis. In this study, we elevated cellular Cer levels in human colon-carcinoma (HT29rev) cells by incubating the cells in the presence of bacterial sphingomyelinase (bSMase) or, alternatively, in the presence of C2-Cer, a short-chain analogue of the sphingolipid. bSMase treatment did not induce apoptosis in these cells, as revealed by a lack of both DNA fragmentation and cleavage of poly(ADP-ribose)polymerase. In contrast, apoptosis did occur upon addition of C2-Cer. These findings led us to study whether differences in the metabolic fate of the excess of Cer, as generated by both treatments, contributed to the observed difference in apoptosis-inducing capacity. C2-Cer was rapidly taken up by HT29rev cells and accumulated due to the absence of substantial metabolic conversion. Upon addition of bSMase, hydrolysis of sphingomyelin resulted in a reduction of that pool to 20% compared with control values, accompanied by a multi-fold increase in Cer level. In spite of the continuous presence of active bSMase, the Cer increase turned out to be transient. Cer levels reached their maximum 1–2 h after addition of bSMase, followed by a significant decrease. Excessive Cer was mainly turned over via cerebrosides into complex glycolipids, including gangliosides. In the presence of glucosylceramide synthase- and/or ceramidase inhibitors, this conversion was significantly blocked and bSMase-generated Cer accumulated in the cells. However, even under these conditions apoptosis did not occur. In conclusion, the inability of bSMase to induce apoptosis of HT29rev cells does not appear to be due to rapid metabolic conversion of excessive Cer. Since apoptosis is induced upon addition of C2-Cer, we therefore propose that the intracellular target involved in the propagation of the apoptotic signal is reached by C2-Cer, but not by bSMase-generated Cer.
Mucus glycoproteins from pig gastric mucosa: different mucins are produced by the surface epithelium and the glands
Authors: NORDMAN Henrik , DAVIES Julia R. , CARLSTEDT Ingemar 
Published: August 2015

An antibody (PGM2B) recognizing a pig gastric-mucin apoprotein reacts with the surface epithelium of pig gastric mucosa. Virtually no reactivity was observed over the mucin-producing cells in the glands, which were recognized by the GlcNAc-selective Griffonia simplicifoliaII (GSA-II) lectin. Mucins from the glandular tissue of the cardiac region, corpus and antrum were purified using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. In the cardiac region, two major mucin populations at 1.5 and 1.4 g/ml were identified. The high-density population reacted preferentially with the PGM2B antibody and resembled mucins from the surface epithelium of this region, whereas the low-density population reacted strongly with the GSA-II lectin and appeared to originate from the glands. In the glandular tissue of corpus, a component with strong GSA-II lectin reactivity, which was distinctly different from the surface mucins from this region, was found at 1.4 g/ml, thus resembling the gland component from the cardiac region. Mucins from antrum glandular tissue contained at least two GSA-II lectin-reactive populations banding at 1.5 and 1.4 g/ml, respectively. Gland mucins from all regions were large oligomeric glycoproteins and heterogeneous with respect to charge properties, as shown by using rate-zonal centrifugation and ion-exchange HPLC, respectively. Gel chromatography of mucin glycopeptides showed that gland mucins from antrum and corpus contained significantly longer glycosylated domains than those from the surface mucosa. Thus, mucins from pig gastric glandular tissue comprise a number of large and oligomeric glycoproteins that differ from those from the surface epithelium in buoyant density, apoprotein structure and carbohydrate substitution.