bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammation-induced changes in expression and glycosylation of genetic variants of α1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice
Authors: van Dijk W , Pos O , van der Stelt M E , Moshage H J , Yap S H , Dente L , Baumann P , Eap C B
Published: August 2015

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.
Expression of cloned human lactoferrin in baby-hamster kidney cells
Authors: Stowell K M , Rado T A , Funk W D , Tweedie J W
Published: August 2015

Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin.
Natural human interferon-α2 is O-glycosylated
Authors: Adolf G R , Kalsner I , Ahorn H , Maurer-Fogy I , Cantell K
Published: August 2015

Natural human interferon alpha 2 (IFN-alpha 2) was isolated from a preparation of partially purified human leucocyte IFN by monoclonal-antibody immunoaffinity chromatography. The purified protein had a specific activity of 1.5 x 10(8) i.u./mg; it was estimated to constitute 10-20% of the total antiviral activity of leucocyte IFN. N-Terminal amino-acid-sequence analysis identified the subspecies IFN-alpha 2b and/or IFN-alpha 2c, whereas IFN-alpha 2a was not detectable. The structure of natural IFN-alpha 2 was found to differ from that of its recombinant (Escherichia coli-derived) equivalent. First, reverse-phase h.p.l.c. showed that natural IFN-alpha 2 was significantly more hydrophilic then expected. Secondly, the apparent molecular mass of the natural protein determined by SDS/PAGE was higher than that of recombinant IFN-alpha 2; incubation under mild alkaline conditions known to eliminate O-linked carbohydrates resulted in a reduction of the apparent molecular mass to that of the recombinant protein. On sequence analysis of proteolytic peptides, Thr-106 was found to be modified. These results suggested that Thr-106 of natural IFN-alpha 2 carries O-linked carbohydrates. Reverse-phase h.p.l.c. as well as SDS/PAGE of natural IFN-alpha 2 showed that glycosylation is heterogeneous. For characterization of the carbohydrate moieties, the protein was treated with neuraminidase and/or O-glycanase and analysed by gel electrophoresis; in addition, glycopeptides obtained by proteinase digestion and separated by h.p.l.c. were characterized by sequence analysis and m.s. Further information on the composition of the glycans was obtained by monosaccharide analysis. The results indicate that natural IFN-alpha 2 contains the disaccharide galactosyl-N-acetylgalactosamine (Gal-GalNAc) linked to Thr-106. In part of the molecules, this core carbohydrate carries (alpha-)N-acetylneuraminic acid, whereas a disaccharide, probably N-acetyl-lactosamine, is bound to Gal-GalNAc in another proportion of the protein. Further glycosylation isomers are present in small amounts. As IFN-alpha 2 is the only IFN-alpha species with a threonine residue at position 106, it may represent the only O-glycosylated human IFN-alpha protein.
Role of serine biosynthesis and its utilization in the alternative pathway from glucose to glycogen during the response to insulin in cultured foetal-rat hepatocytes
Authors: Bismut H , Plas C
Published: August 2015

The role of serine as a possible intermediate of the alternative pathway from glucose to glycogen was investigated under basal and insulin-stimulated conditions in 18-day cultured foetal-rat hepatocytes because these cells cannot use pyruvate-derived metabolites [Bismut & Plas (1989) Biochem. J. 263, 889-895]. Incubation of cells with [U-14C]glucose for 24 h led to a release of labelled serine in the medium concomitantly with a net serine production (100 nmol/24 h per culture). The rate of [14C]serine formation (close to 3 nmol/h per culture) indicated that a large part of newly formed serine originated from glucose. When short-term experiments were performed at day 2, glycogen labelling from [U-14C]serine or [U-14C]glycine, which was increased 3-fold by insulin after 2 h, evidenced their participation as glycogenic precursors. When a double-isotope procedure with [U-14C,3-3H]glucose was used, the direct and the alternative pathways from glucose were found to contribute to glycogenesis by 75 and 25% respectively. Cycloserine (18 mM), a transaminase inhibitor, strongly inhibited glycogen labelling from [U-14C] serine while producing a 70% increase in glucose incorporation by the alternative pathway, in both the presence and the absence of insulin. The inhibitor had no effect on the direct pathway from glucose to glycogen. Supplementation with 1 mM-hydroxypyruvate, a serine-derived metabolite, did not affect direct glucose incorporation, whereas the alternative pathway was stimulated whether insulin was present or not. These results indicate that the sequence glucose----serine----glycogen is operative in cultured foetal hepatocytes. The alternative pathway interferes with hydroxypyruvate utilization, and is likely mediated by the serine aminotransferase pathway, independently of the acute glycogenic action of insulin.
Characterization of phospholipase A2 in monocytic cell lines. Functional and biochemical aspects of membrane association
Authors: Rehfeldt W , Hass R , Goppelt-Struebe M
Published: August 2015

Phospholipase A2 activity was characterized in the human monocytic tumour-cell lines U937 and THP1. The enzyme showed an alkaline pH optimum and substrate specificity for arachidonoyl-phosphatidylcholine. The activation of phospholipase A2 required bivalent cations (Ca2+ greater than Mg2+ = Sr2+ greater than Ba2+). Investigation of the subcellular distribution of the enzyme revealed that the phospholipase A2 activity was shifted to the cytosol in the presence of EDTA, indicating that the association of the enzyme with the cellular membranes is Ca2+ (bivalent-cation)-dependent. Stimulation of THP1 cells for 2-4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) activated cytosolic and membrane-bound phospholipase A2. At this time, no effect of PMA on phospholipase A2 activity was observed in the less mature U937 cells. However, when both cell lines were induced to differentiate along the monocytic pathway by a 2-3-day treatment with PMA, the cells released significant amounts of arachidonic acid and prostanoids. Compared with undifferentiated control cells, these PMA-differentiated cells showed a decrease in cytosolic phospholipase A2 activity and an increase in membrane-bound activity. Membrane-bound and cytosolic enzyme showed the same pH optimum, Ca(2+)-dependency and substrate specificity. These data indicate that membrane-bound and cytosolic phospholipase A2 activities represent one enzyme and that the membrane-bound form is the biologically active phospholipase A2.
Heterogeneity of mucus glycoproteins from cystic fibrotic sputum. Are there different families of mucins?
Authors: Thornton D J , Sheehan J K , Carlstedt I
Published: August 2015

High-Mr mucin glycopeptides prepared from sputum of an individual with cystic fibrosis (CF) were studied by ion-exchange h.p.l.c. The glycopeptides were heterogeneous and a number of partially resolved populations were identified. Whole mucins from the gel phase were separated into four fractions by isopycnic density-gradient centrifugation in CsCl, and high-Mr glycopeptides from these fractions were examined by ion-exchange h.p.l.c. The acidic nature of the high-Mr glycopeptides increased with increasing buoyant density of the intact mucins, and a periodate-Schiff (PAS)-rich and an extremely high-iron diamine (HID)-reactive component were present in the lowest and highest density fractions respectively. The various glycopeptide populations were identified in different proportions in mucins from four other individuals with CF. CF sputum thus seems to contain distinct mucin populations containing different oligosaccharide clusters corresponding to these high-Mr glycopeptides.
Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site
Authors: Legrand D , Mazurier J , Maes P , Rochard E , Montreuil J , Spik G
Published: August 2015

Labelling of human lactotransferrin with fluorescein 5′-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.
Transport of taurine and its regulation by protein kinase C in the JAR human placental choriocarcinoma cell line
Authors: Kulanthaivel P , Cool D R , Ramamoorthy S , Mahesh V B , Leibach F H , Ganapathy V
Published: August 2015

The JAR human placental choriocarcinoma cell line transports taurine, concentrating it over 1000-fold inside the cell. The transport system is energized by a Na+ gradient and exhibits an absolute requirement for Cl-. Neutral beta-amino acids such as beta-alanine and hypotaurine effectively compete with the system, whereas neutral alpha-amino acids such as alanine, leucine and alpha-aminoisobutyric acid do not. The transport system interacts with gamma-aminobutyric acid to an appreciable extent. Kinetic analysis reveals that the taurine transport system in this cell line is of a high-affinity and low-capacity type (apparent dissociation constant 2.3 +/- 0.3 microM; maximal velocity 88.5 +/- 5.0 pmol/3 min per mg of protein). Pretreatment of the JAR choriocarcinoma cells with phorbol 12-myristate 13-acetate results in the inhibition of the taurine transport system in a dose-dependent manner. The inhibition is blocked by co-treatment of the cells with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, has no effect on the transport system. These data show that the choriocarcinoma cells express a taurine transporter with characteristics similar to those of the taurine transporter described in the normal human placenta, and that the activity of the transporter in these cells is under the regulatory control of protein kinase C.
Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture
Authors: Thomas G J , Mason R M , Davies M
Published: August 2015

1. The newly synthesized proteoglycans from human adult glomerular mesangial cells labelled in vitro for 24 h with [35S]sulphate have been characterized using biochemical and immunological techniques. 2. The following proteoglycans were identified (% of total synthesized). (i) A large chondroitin sulphate proteoglycan, CSPG-I, Mr approximately 1 x 10(6) (10.6%). This proteoglycan consisted of a protein core of Mr approximately 4 x 10(5) and glycosaminoglycan chains of Mr 2.5 x 10(4), and was present in both the cell layer and the culture medium. (ii) A major small dermatan sulphate proteoglycan, DSPG-I, Mr 3.5 x 10(5) (46%), which was mainly located in the culture medium. (iii) A second minor small dermatan sulphate, DSPG-II, Mr approximately 2 x 10(5) (9.8%). This molecule was exclusively located in the culture medium. (iv) A large heparan sulphate proteoglycan, HSPG-I, Mr 8 x 10(5) (3.3%). (v) A second large heparan sulphate proteoglycan HSPG-II, Mr approximately 6 x 10(5) (23%). HSPG-I and HSPG-II were extracted from both the culture medium and the cell layer. 3. Western blot analysis of the core proteins released by chondroitin ABC lyase treatment of DSPG-I and DSPG-II identified these dermatan sulphate proteoglycans as biglycan and decorin respectively. Both DSPG-I and DSPG-II had core proteins of Mr 45,000. 4. The cell-layer-associated forms of CSPG-I, HSPG-I and HSPG-II were accessible to limited trypsin treatment, bound to octyl-Sepharose and could be inserted into liposomes, indicating a possible cell membrane location. 5. Pulse-chase experiments indicated that the cell-layer-associated [35S]proteoglycans undergo limited metabolism to inorganic [35S]sulphate, the majority of which is accounted for by the degradation of HSPG-II and to a lesser extent DSPG-I.
A large accumulation of non-muscle myosin occurs at first entry into M phase in rat vascular smooth-muscle cells
Authors: Grainger D J , Hesketh T R , Metcalfe J C , Weissberg P L
Published: August 2015

Vascular smooth-muscle cells (VSMCs) from rat aortae contained very little non-muscle myosin heavy chain (MHC) immediately after dispersal, and the protein did not accumulate if the cells were held in G0/G1 phase by withholding serum or were held in first S phase by the addition of bromodeoxyuridine (BrdU). However, non-muscle MHC accumulated by greater than 20-fold per cell during first M phase, when over 80% of the cells divided between 48 h and 72 h after addition of serum. Delaying the addition of serum caused a delay in the accumulation of the non-muscle MHC until the cells subsequently entered M phase. If the cells were held in M phase at the metaphase/anaphase boundary by nocadazole, the accumulation of non-muscle myosin still occurred, although division was blocked. When the cells were pulse-labelled with [35S]methionine, it was found that non-muscle MHC was one of the major proteins being made and that its synthesis occurred at similar rates throughout the cell cycle. This implied that the rate of degradation of the protein before first M phase was much faster than in M phase, when the protein accumulated rapidly. This was confirmed by direct measurements of the rate at which [35S]methionine-labelled non-muscle MHC disappeared from the cells, which gave a half-life for the protein of about 8 h before M phase but about 5 days in post-mitotic cells, i.e. an increase of approx. 15-fold. These data are consistent with the hypothesis that there is a mechanism in VSMCs which shortens the half-life of the protein before first M phase and that the accumulation of non-muscle MHC which results from the increase in half-life at first M phase may be necessary for division of these cells.
Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride
Authors: Ma Y Z , Tsou C L
Published: August 2015

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.
Failure of [32P]ADP-ribosylation by pertussis toxin to determine Giα content in membranes from various human tissues. Improved radioimmunological quantification using the 125I-labelled C-terminal decapeptide of retinal transducin
Authors: Böhm M , Larisch K , Erdmann E , Camps M , Jakobs K , Gierschik P
Published: August 2015

The quantitative determination of pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G-proteins) in cell membranes is still a problem. Pertussis-toxin-catalysed [32P]ADP-ribosylation strongly relies on the substrate quality of the alpha-subunits and is influenced by the concentration of nucleotides, beta gamma-subunits, the physicochemical properties of the membranes influencing the availability of Gi alpha for pertussis toxin, and covalent modification of Gi alpha. Quantification of immunoreactive material on Western blots can be only imprecisely performed by two-dimensional densitometry. In order to generate a method for quantification of pertussis-toxin-sensitive G-proteins in membranes we have developed a fast and sensitive radioimmunoassay. The C-terminal decapeptide of retinal transducin alpha (KENLKDCGLF) was 125I-labelled and used as tracer. Polyclonal antiserum (DS 4) was raised against this peptide. Gi alpha proteins were determined by competition of solubilized membranes for 125I-KENLKDCGLF binding to DS 4 using dilutions of retinal transducin alpha as standard. The interassay variation was less than 10%, with a sensitivity of 2.5 micrograms/ml. The density of Gi alpha was highest in human adipose tissue, followed by HL60 cells, lung, mononuclear leucocytes, thrombocytes and left ventricular myocardium. A striking difference was observed between the density of Gi alpha and the amount of incorporation of [32P]ADP-ribose into the 40 kDa membrane proteins by pertussis toxin in the same samples. This is also demonstrated by comparison of the weak [32P]ATP-ribosylation of pertussis toxin substrates with the density of immunoreactive Gi alpha on Western blots in tissues such as lung. This study shows that the Gi alpha content can be exactly determined by a sensitive and fast radioimmunoassay using iodinated synthetic peptide homologues of Gi alpha proteins. Radioimmunological quantification of Gi alpha might be able to detect the ‘true’ Gi alpha content of membranes without being hampered by influences on the [32P]ADP-ribosylation reaction. It is concluded that this newly developed method may become an important tool for studying expression of Gi alpha proteins in a variety of tissues or cell types, and for precisely quantifying the changes caused by pathological conditions.
A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom
Authors: Huang T F , Sheu J R , Teng C M
Published: August 2015

The interaction of fibrinogen with its receptors on platelet surfaces leads to platelet aggregation. A snake-venom peptide, trigramin, has previously been demonstrated to inhibit platelet aggregation by acting as a fibrinogen-receptor antagonist. By means of gel filtration, ionic-exchange chromatography and reverse-phase h.p.l.c., a potent platelet-aggregation inhibitor, triflavin, has now been purified from the venom of Trimeresurus flavoviridis. The purified triflavin is a single-chain polypeptide, consisting of about 71 amino acid residues with a molecular mass of 7600 Da, and its N-terminal sequence is Gly-Glu-Glu-Cys-Asp. Triflavin dose-dependently inhibited human platelet aggregation stimulated by ADP, adrenaline, collagen, thrombin or prostaglandin endoperoxide analogue U46619 in preparations of platelet-rich plasma, platelet suspension and whole blood. Its IC50 ranged from 38 to 84 nM, depending on the aggregation inducer used and the platelet preparation. However, triflavin apparently did not affect the platelet shape change and ATP-release reactions caused by these agonists. Triflavin inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner, indicating that it directly interferes with the binding of fibrinogen to its receptors on platelet membranes exposed by elastase treatment. Additionally, triflavin dose-dependently blocked 125I-labelled fibrinogen binding to ADP-activated platelets. In conclusion, triflavin inhibits platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors on platelet membranes.
Bacterial lipopolysaccharide-stimulated GTPase activity in RAW 264.7 macrophage membranes
Authors: Tanke T , van de Loo J W , Rhim H , Leventhal P S , Proctor R A , Bertics P J
Published: August 2015

The molecular mechanisms surrounding the toxicity and high mortality rate that accompany the release of bacterial lipopolysaccharide (LPS) are unclear, although its potent activity suggests that an amplification system is involved. Because previous studies suggest that a guanine-nucleotide-binding protein (G-protein) may participate in LPS action, we have evaluated the effects of LPS on GTPase activity in membranes isolated from macrophage (RAW 264.7) and fibroblast (B82L) cell lines. LPS induced substantial GTPase activation (200-300% above basal), and kinetic analyses indicated that the maximal LPS-stimulated increase in velocity is observed within 15 min, that it is a low-Km (for GTP) activity, that it can be enhanced by ammonium sulphate, and that it appears to be pertussis toxin-insensitive. Moreover, the LPS-enhanced GTPase activity was not antagonized by phosphatase/ATPase inhibitors such as p-nitrophenyl phosphate, ouabain, bafilomycin or N-ethylmaleimide, and in fact was potentiated by the addition of ATP or ADP. Conversely, the LPS precursor, lipid X, which can decrease the lethal effects of LPS, was found to dose-dependently inhibit the LPS-mediated stimulation of GTPase activity. Half-maximal inhibition was seen at the same lipid X/LPS ratio known to be effective in vivo, i.e. 1:1(w/w). These effects appear to be specific because other phospholipids, detergents and glycosides neither stimulated basal, nor inhibited LPS-induced, GTPase activity. These data suggest the involvement of a GTPase in LPS action, and indicate that lipid X may act to directly antagonize LPS at this level.
Down-regulation of mannose receptors on macrophages after infection with Leishmania donovani
Authors: Basu N , Sett R , Das P K
Published: August 2015

Macrophages express a mannose-specific endocytosis receptor that binds and internalizes mannose-terminated glycoproteins. Infection of mouse peritoneal macrophages with Leishmania donovani resulted in a decrease in mannose-receptor activity. With 125I-labelled beta-glucuronidase as ligand, a 2-fold decrease in uptake rate was observed in infected cells, with no change in Kuptake. Cell-surface binding of 125I-mannose-BSA was diminished 2.5-fold after infection. The decrease in ligand binding appeared to be due to a decrease in the number of sites, with no change in affinity. Elimination of parasites from infected cells by treatment with neoglycoprotein-conjugated methotrexate resulted in an increase in receptor number. Cycloheximide suppressed the drug-treatment-mediated rise in receptor number in infected macrophages. A decrease in receptor activity was also observed in liver Kupffer cells isolated from parasite-infected mice. Binding of ligand by another carbohydrate receptor, the mannose 6-phosphate receptor, was not altered by infection. Phagocytosis of yeast cells was also not altered. These results suggest that mannose receptor synthesis in macrophages is specifically suppressed after infection with Leishmania parasites.
Heterogeneous modulation of acute-phase-reactant mRNA levels by interleukin-1 β and interleukin-6 in the human hepatoma cell line PLC/PRF/5
Authors: Steel D M , Whitehead A S
Published: August 2015

The acute-phase response to tissue injury and inflammation is accompanied by a dramatic increase in the hepatic synthesis of plasma proteins known as acute-phase reactants (APRs). This response is mediated by cytokines produced in part by activated macrophages at the site of inflammation; glucocorticoids have also been implicated as playing a regulatory role. The effects of the cytokines interleukin (IL)-1 beta and -6, alone or in combination, and in the absence or presence of the synthetic glucocorticoid dexamethasone, on the levels of APR mRNAs in the human hepatoma cell line PLC/PRF/5 were analysed. The accumulation of APR mRNAs [the complement components C3, factor B and Cl inhibitor; the major APRs C-reactive protein (CRP) and serum amyloid A protein and the CRP analogue serum amyloid P protein] was determined in dose-response and time-course studies. The APRs differed from each other in their responses to IL-1 beta alone, IL-6 alone, and IL-1 beta plus IL-6. Dexamethasone enhanced the cytokine-driven induction of a subset of APR mRNAs. These studies detail the heterogeneity of the ‘in vitro’ acute-phase response to defined mediators.
The overlapping octamer/TAATGARAT motif is a high-affinity binding site for the cellular transcription factors Oct-1 and Oct-2
Authors: Dent C L , Latchman D S
Published: August 2015

The octamer motif in cellular promoters and the related TAATGARAT element in the herpes simplex virus (HSV) immediate-early promoters can both bind cellular octamer-binding proteins. The overlapping octamer/TAATGARAT elements (consensus ATGCTAATGARAT) found in the HSV-1 IE1 promoter thus represent a composite motif, each portion of which can independently bind octamer-binding protein. By comparing the binding characteristics of this composite motif with its individual elements, we show that it binds a single molecule of either Oct-1 or Oct-2 with much higher affinity than does either an octamer or TAATGARAT motif alone. This strong binding allows this element to direct a much higher level of gene expression when linked to a heterologous promoter than that observed with each of its individual components.
Dose-dependent acinar induction of cytochromes P450 in rat liver. Evidence for a differential mechanism of induction of P450IA1 by β-naphthoflavone and dioxin
Authors: Bars R G , Elcombe C R
Published: August 2015

Rats received various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Aroclor 1254 (ARO), beta-naphthoflavone (BNF) or phenobarbital (PB), and the hepatic expression of cytochromes P450IA1 and/or P450IIB1/IIB2 was analysed by immunohistochemistry and Western blotting. A clear heterogeneous acinar induction of IA1 was detected when a low dose of TCDD, ARO or BNF was administered. When a low dose of TCDD or ARO was administered, IA1 was found to be induced primarily in hepatocytes located in acinus zone 3, whereas when a low dose of BNF was administered, IA1 was found to be preferentially induced in hepatocytes located in acinus zone 1. A clear zonal induction of IIB1/IIB2 was also observed when a low dose of PB or ARO was administered. Both compounds induced IIB1/IIB2 preferentially in hepatocytes located in acinus zone 3. When rats were administered high doses of TCDD, ARO, BNF or PB there was no zonal pattern of induction of IA1 or IIB1/IIB2; instead, a pan-acinar induction of these enzymes was observed. These results indicate that the overall hepatic concentration of IA1 or IIB1/IIB2 is merely dependent on the proportion of ‘induced hepatocytes’ within the acinus, which in turn depends on the dose of the inducer.
Monoclonal antibodies specific for the C-terminus of the laminin B2 subunit. Evidence for glycosylation differences between murine and human laminin
Authors: Brown J C , Spragg J H , Taylor P W
Published: August 2015

We have raised a panel of monoclonal antibodies against a beta-galactosidase fusion protein (XLB2.1) containing the C-terminal 153 amino acids of the murine laminin B2 subunit. Five of the nine antibodies characterized recognize human placental laminin as well as murine Engelbreth-Holm-Swarm (EHS)-tumour laminin. Only two of the antibodies recognize both rat parietal-yolk-sac laminin and murine EHS-tumour laminin. Two antibodies recognize an epitope on the human laminin B2 subunit which is masked by N-linked oligosaccharide in murine EHS-tumour laminin. These antibodies also fail to bind to laminin from adult-mouse tissues. These results demonstrate a species-specific difference in the glycosylation of the laminin B2 subunit.
The substrate-specificity of human lysosomal α-d-mannosidase in relation to genetic α-mannosidosis
Authors: al Daher S , de Gasperi R , Daniel P , Hall N , Warren C D , Winchester B
Published: August 2015

The specificity of human liver lysosomal alpha-mannosidase (EC 3.2.1.24) towards a series of oligosaccharide substrates derived from high-mannose, complex and hybrid asparagine-linked glycans and from the storage products in alpha-mannosidosis was investigated. The enzyme hydrolyses all alpha(1-2)-, alpha(1-3)- and alpha(1-6)-mannosidic linkages in these glycans without a requirement for added Zn2+, albeit at different rates. A major finding of this study is that all the substrates are hydrolysed by non-random pathways. These pathways were established by determining the structures of intermediates in the digestion mixtures by a combination of h.p.t.l.c. and h.p.l.c. before and after acetolysis. The catabolic pathway for a particular substrate appears to be determined by its structure, raising the possibility that degradation occurs by an uninterrupted sequence of steps within one active site. The structures of the digestion intermediates are compared with the published structures of the storage products in mannosidosis and of intact asparagine-linked glycans. Most but not all of the digestion intermediates derived from high-mannose glycans have structures found in intact asparagine-linked glycans of human glycoproteins or among the storage products in the urine of patients with mannosidosis. However, the relative abundances of these structures suggests that the catabolic pathway is not the same as the processing pathway. In contrast, the intermediates formed from the digestion of oligosaccharides derived from hybrid and complex N-glycans are completely different from any processing intermediates and also from the oligosaccharides of composition Man2-4GlcNAc that account for 80-90% of the storage products in alpha-mannosidosis. It is postulated that the structures of these major storage products arise from the action of an exo/endo-alpha(1-6)-mannosidase on the partially catabolized oligomannosides that accumulate in the absence of the main lysosomal alpha-mannosidase.
Glycosylation, activity and secretion of lipoprotein lipase in cultured brown adipocytes of newborn mice. Effect of tunicamycin, monensin, 1-deoxymannojirimycin and swainsonine
Authors: Masuno H , Schultz C J , Park J W , Blanchette-Mackie E J , Mateo C , Scow R O
Published: August 2015

The effect of inhibitors on the glycosylation, activity and secretion of lipoprotein lipase was studied in brown adipocytes cultured from newborn mice. Such cells synthesized and secreted active lipoprotein lipase. It is generally accepted that active lipoprotein lipase is a homodimer. Glycosylation of lipoprotein lipase was analysed by PAGE of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase immunoprecipitated from cells incubated for 1-2 h with [35S]methionine. The most prevalent 35S-labelled lipase subunit (Mr 57,000-58,000) in these cells contained endo H-resistant oligosaccharide chains, the next most prevalent contained totally endo H-sensitive chains, and the least prevalent subunit contained partially endo H-sensitive chains. Complete blocking of the glycosylation of lipoprotein lipase with tunicamycin (1 microgram/ml) for 24 h resulted in synthesis of an inactive non-secretable form of lipase with a smaller subunit (Mr 51,000-52,000). Immunofluorescent studies showed that unglycosylated lipase in tunicamycin-treated cells was retained in the endoplasmic reticulum. Cells treated with 1 microM-monensin, an intra-Golgi transport inhibitor, synthesized an active form of lipase which was not secreted, but was retained in the Golgi. The lipase in monensin-treated cells contained only partially or totally endo H-sensitive chains. Blocking either Golgi mannosidase I with 4 mM-1-deoxymannojirimycin or Golgi mannosidase II with 10 microM-swainsonine resulted in production of a form of lipoprotein lipase which was active and secreted, and which contained only endo H-sensitive chains. Our findings demonstrate that core glycosylation of lipoprotein lipase in the endoplasmic reticulum is required for lipase activity and transport from the reticulum, whereas processing of the oligosaccharide chains to endo H-resistant (complex) type chains in the Golgi is not required for either the activity or the secretion of lipoprotein lipase.
Heterogeneity of rat liver cytosol casein kinase 2. Association between the α/α' -subunits of casein kinase 2 and the phosphorylatable protein pp49
Authors: Molina E , Plana M , Itarte E
Published: August 2015

Casein kinase 2 activity could be resolved into three peaks by chromatography on DEAE-Sepharose. The peak eluted at high salt concentrations (casein kinase 2b) showed molecular and kinetic properties typical of the heterotetramer composed of alpha-(or alpha'-) and beta-subunits. In contrast, the peak that was eluted at low salt concentrations (casein kinase 2a) contained no beta-subunit but a phosphorylatable protein of 49 kDa (pp49), in addition to the alpha/alpha'-subunits. The presence of alpha/alpha'/alpha"-subunits in preparations of casein kinases 2a and 2b was confirmed by immunological assays. Casein kinase 2a had low specific activity and a very high apparent Km for beta-casein. The peak eluted at intermediate ionic strength contained the alpha/alpha'-subunits and variable amounts of beta-subunit and pp49, and had kinetic properties intermediate between those of casein kinases 2a and 2b. Experiments based on heat inactivation, inhibition by low concentrations of heparin and ability to use GTP as substrate suggested that phosphorylation of pp49 was catalysed by the alpha/alpha'-subunits of casein kinase 2. No similarities were observed in the phosphopeptide maps of pp49 and beta-subunit. These results show that the alpha/alpha'-subunits of rat liver cytosol casein kinase 2 can form complexes not only with the beta-subunit but also with pp49, and that the complexes containing pp49 have a reduced affinity for the exogenous protein substrate beta-casein.
Ethoxyquin-induced resistance to aflatoxin B1 in the rat is associated with the expression of a novel Alpha-class glutathione S-transferase subunit, Yc2, which possesses high catalytic activity for aflatoxin B1-8,9-epoxide
Authors: Hayes J D , Judah D J , McLellan L I , Kerr L A , Peacock S D , Neal G E
Published: August 2015

A purification scheme has been devised for two ethoxyquin-inducible Alpha-class glutathione S-transferases (GSTs) which possess at least 25-fold greater activity towards aflatoxin B1 (AFB1)-8,9-epoxide than that exhibited by the GSTs (i.e. F, L, B and AA) that have been described previously. These two enzymes are both heterodimers and both contain a subunit of Mr 25,800. This subunit has been isolated from both of the GST isoenzymes and, after cleavage with CNBr, it has been subjected to automated amino acid sequencing. The primary structure of the Mr 25,800 subunit revealed that it forms part of a subfamily of Alpha-class GSTs which possess closest identity (about 92%) with the Yc subunit of apparent Mr 27,500, which is encoded by the recombinant cDNA clone pGTB42 [Telakowski-Hopkins, Rodkey, Bennett, Lu & Pickett (1985) J. Biol. Chem. 260, 5820-5825]. As these two GSTs possess less than 70% sequence identity with the Ya1 and Ya2 subunits, both of Mr 25,500, the constitutively expressed Yc subunit of Mr 27,500 has been renamed Yc1 and the ethoxyquin-inducible GST of Mr 25,800 has been designated Yc2. Using this nomenclature, the two GSTs with high activity for AFB1-8,9-epoxide are Ya1Yc2 and Yc1Yc2. Although evidence suggests that induction of Yc2 is responsible for the high detoxification capacity of livers from ethoxyquin-treated rats for AFB1-8,9-epoxide, resistance towards AFB1 may be multifactorial in this instance as dietary ethoxyquin also induces the Ya1, Ya2 and Yc1 subunits about 2.2-, 10.9- and 2.7-fold respectively. Besides the induction of GST by ethoxyquin, activity towards AFB1-8,9-epoxide is also elevated in the livers of neonatal rats and in livers that contain preneoplastic nodules. Western blotting experiments show that Yc2 is not present in hepatic cytosol from adult rats fed on normal diets but is expressed in neonatal rat livers and in the livers of adult rats that contain preneoplastic nodules that have arisen as a consequence of consuming diets contaminated with AFB1.
Verapamil prevents the effects of daunomycin on the thermotropic phase transition of model lipid bilayers
Authors: Canaves J M , Ferragut J A , Gonzalez-Ros J M
Published: August 2015

High-sensitivity differential scanning calorimetry and fluorescence-depolarization techniques were used to study how the presence of daunomycin and/or verapamil affect the thermotropic behaviour of dipalmitoyl phosphatidylcholine (DPPC) vesicles. Daunomycin, a potent anti-cancer agent, perturbs the thermodynamic parameters associated with the lipid phase transition: it decreases the enthalpy change, lowers the transition temperature and reduces the co-operative behavior of the phospholipid molecules. Verapamil, on the other hand, produces smaller alterations in the lipid phase transition. However, when daunomycin and verapamil are present simultaneously in the DPPC vesicles, it is observed that verapamil prevents, in a concentration-dependent manner, the alteration in the phospholipid phase transition expected from the presence of daunomycin in the bilayer. Furthermore, drug-binding studies suggest that the observed interference of verapamil in the daunomycin/phospholipid interaction occurs without a decrease in the amount of daunomycin bound to the lipid bilayer and without the formation of a daunomycin-verapamil complex. Because of the importance of drug-membrane interactions in anthracycline cytotoxicity, we speculate that the lipid bilayer of biological membranes may provide appropriate sites at which the presence of verapamil influences the activity of daunomycin.
Phosphorylation of a cell surface 112 kDa protein by an ecto-protein kinase in rat L6 myoblasts
Authors: Chen X Y , Lo T C Y
Published: August 2015

Studies with subconfluent day 2 cultures of rat myoblasts revealed that a cell surface 112 kDa protein could be phosphorylated by extracellular ATP. Analysis of the phosphorylated 112 kDa protein suggested the involvement of a serine protein kinase. The following evidence indicated the cell surface location of this protein kinase: (i) extracellular ATP was unable to penetrate the cell membrane under our experimental conditions; (ii) the phosphorylated protein profile of intact cells differed significantly from that of broken cells; (iii) the phosphorylation of the 112 kDa protein could be abolished by pretreatment of cells with very low concentrations of trypsin; (iv) the phosphorylated 112 kDa protein could be dephosphorylated by exogenously added alkaline phosphatase; (v) the phosphorylation of the 112 kDa protein was inhibited by exogenously added proteins; and (vi) exogenously added proteins could be phosphorylated by intact cells under similar experimental conditions. The phosphorylated 112 kDa protein was detected only when the reaction was carried out in the presence of Ca2+, Mg2+, and F- ions. Kinetic analysis that revealed that the Km value of the ecto-protein kinase for ATP was 0.04 microM, and the Vmax. value for phosphorylation of the 112 kDa protein was 1.67 x 10(-4) pmol/min per 10(5) cells. Data presented in the accompanying paper [Chen & Lo (1991) Biochem. J. 279, 475-482] show that there was a constant and adequate supply of ATP on the cell surface of rat myoblasts for efficient functioning of this protein kinase, and that mutants defective in either the ecto-protein kinase or the 112 kDa protein were also impaired in myogenic differentiation. This and other biochemical studies suggest that the ecto-protein kinase and the 112 kDa protein might play important roles in myogenic differentiation.
Factors affecting the ability of glycosylphosphatidylinositol-specific phospholipase D to degrade the membrane anchors of cell surface proteins
Authors: Low M G , Huang K S
Published: August 2015

Mammalian serum and plasma contain high levels of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). Previous studies with crude serum or partially purified GPI-PLD have shown that this enzyme is capable of degrading the GPI anchor of several purified detergent-solubilized cell surface proteins yet is unable to act on GPI-anchored proteins located in intact cells. Treatment of intact ROS17/2.8, WISH or HeLa cells (or membrane fractions prepared from them) with GPI-PLD purified from bovine serum by immunoaffinity chromatography gave no detectable release of alkaline phosphatase into the medium. However, when membranes were treated with GPI-PLD in the presence of 0.1% Nonidet P-40 substantial GPI anchor degradation (as measured by Triton X-114 phase separation) was observed. The mechanism of this stimulatory effect of detergent was further investigated using [3H]myristate-labelled variant surface glycoprotein and human placental alkaline phosphatase reconstituted into phospholipid vesicles. As with the cell membranes the reconstituted substrates exhibited marked resistance to the action of purified GPI-PLD which could be overcome by the inclusion of Nonidet P-40. Similar results were obtained when crude bovine serum was used as the source of GPI-PLD. These data indicate that the resistance of cell membranes to the action of GPI-PLD is not entirely due to the action of serum or membrane-associated inhibitory factors. A more likely explanation is that, in common with many other eukaryotic phospholipases, the action of GPI-PLD is restricted by the physical state of the phospholipid bilayer in which the substrates are embedded. These data may account for the ability of endothelial and blood cells to retain GPI-anchored proteins on their surfaces in spite of the high levels of GPI-PLD present in plasma.
Structure–function relationship of the human erythrocyte plasma membrane Ca2+-ATPase revealed by V8 protease treatment
Authors: Wang K K W , Roufogalis B D , Kuo T H
Published: August 2015

Treatment of the solubilized and purified Ca(2+)-translocating ATPase (Ca(2+)-ATPase) (136 kDa) from human erythrocyte plasma membranes with endoproteinase Glu-C from Staphylococcus aureus strain V8 (V8 protease) yielded transient fragments of 96 kDa and 76 kDa and more stable fragments of 60 kDa and 37/36 kDa (doublet). The presence of calmodulin did not alter the fragmentation pattern. The 60 kDa fragment contains the protein kinase C (bovine brain) phosphorylation site(s), which we previously localized in the C-terminal region [Wang, Wright, Machan, Allen, Conigrave & Roufogalis (1991) J. Biol. Chem. 266, 9078-9085]. On the other hand, the 37/36 kDa fragments possess the ability to form an acyl-phosphate intermediate. Furthermore, the presence of the 60 kDa and 37/36 kDa fragments together results in expression of calmodulin-sensitive Ca(2+)-ATPase activity. However, further degradation of the 60 kDa fragment was coupled with the appearance of calmodulin-independent activity, whereas the 37/36 kDa fragment doublet remained stable. It was concluded that the 60 kDa and the 37/36 kDa fragments: (a) together represent the C-terminal two-thirds of the enzyme, which is functional as an Ca(2+)-ATPase, (b) were produced by a single cleavage near the C-terminal side of the cytosolic catalytic domain, and (c) probably remain physically and functionally associated even after cleavage has occurred. At the C-terminus, the basic calmodulin-binding domain is flanked by two highly acidic regions (domains A and B). Our results indicate that domains A and B, despite containing many Asp and Glu residues, were not readily cleaved by V8 protease, which is known to cleave selectively peptide bonds at the C-terminal side of Asp and Glu. However, if the Ca(2+)-ATPase were pre-digested with calpain I from human erythrocytes, which removed its calmodulin-binding domain (along with domain B), multiple cleavages by V8 protease in domain A were then readily observed. We propose that the calmodulin-binding domain is closely associated with the acidic domains A and B and that these acidic domains might help to co-ordinate the stimulation of the enzyme by calmodulin.
Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins
Authors: Adam M , Damblon C , Jamin M , Zorzi W , Dusart V , Galleni M , el Kharroubi A , Piras G , Spratt B G , Keck W
Published: August 2015

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.