bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of enzyme release from human polymorphonuclear leukocytes: further evidence for the independent regulation of granule subpopulations
Authors: Murphy Patricia , Hart David A.
Published: December 2009

Addition of 5–20 mM LiCl to purified human polymorphonuclear leukocytes led to the release of lysozyme, the specific granule constituent, but not the release of elastase which is in azurophilic granules. In contrast, 2.5–10 μg cytochalasin D/mL induced the release of both lysozyme and elastase. Addition of lipopolysaccharide to leukocytes did not induce enzyme release but primed cells for enhanced release induced by cytochalasin D. Lipopolysaccharide also primed cells for enhanced release of lysozyme by either N-formylmethionylleucylphenylalanine (fMLP) or Li+ but did not prime cells for elastase release by these stimuli. In contrast, fMLP + cytochalasin D interacted synergistically, leading to enhanced elastase release but not lysozyme release from the cells. Additional experiments with combinations of secretogogues and lipopolysaccharide yielded results consistent with the hypothesis that specific granules and subpopulations of azurophilic granules are under separate regulation and, thus, may be influenced by separate elements of intracellular second messenger systems.
Reassembly of active 30S ribosomal subunits with an unmethylated in vitro transcribed 16S rRNA
Authors: Melançon Pierre , Gravel Michel , Boileau Guy , Brakier-Gingras Léa
Published: December 2009

A plasmid has been constructed, which contains the 16S ribosomal RNA gene of Escherichia coli immediately downstream from a phage T7 promoter. In vitro transcription of this gene by RNA polymerase of the T7 phage yielded an unmethylated 16S rRNA that could be used for the assembly of functional 30S subunits. These subunits, when assayed in a poly(U)-directed translation assay, were as active as 30S subunits reconstructed with native 16S rRNA. This system opens the possibility of investigating the role of the methylations of the rRNA and the functional consequences of site-directed mutagenesis in a rRNA gene. An application of this system was provided by generating a 16S rRNA transcript shortened by about 30 nucleotides at the 3′ end. This truncated rRNA could be reassembled into 30S subunits, about 70% as active as 30S subunits reconstructed with the full-length 16S rRNA transcript.
Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro
Authors: Lev Anat , Holland Paul C.
Published: December 2009

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin–Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen–Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63 000 – 72 000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.
Identification of a human osteosarcoma-associated glycoprotein with monoclonal antibodies: relationship with alkaline phosphatase
Authors: Nakamura Takashi , Gross Michael , Yamamuro Takao , Liao Shuen-Kuei
Published: December 2009

The molecular nature of an osteosarcoma-associated antigen was investigated with the three monoclonal antibodies Ost6 (immunoglobulin (IgG1), Ost7 (IgG1), and Ost15 (IgG2a), which selectively react with frozen sections of osteosarcoma and chondrosarcoma tissues. When tested with a panel of 41 human cell lines in the mixed hemadsorption assay, the antibodies reacted similarly with three of six osteosarcomas, one choriocarcinoma, one teratoma, and one osteoblast-like culture, but failed to react with 32 lines of normal and other tumor cell types. Immunoprecipitation plus sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis and sequential immunoprecipitation studies revealed that in [35S]methionine- or [l4C]glucosamine-labelled osteosarcoma cells the three antibodies detected a single glycoprotein, with an apparent molecular mass of 86 kilodaltons (kDa), which was not affected by reducing conditions. Tunicamycin treatment and pulse–chase experiments showed glycosylation of this molecule to be N-linked; it arose from a 54-kDa polypeptide precursor. Alkaline phosphatase activity was detected in the material rich in 86-kDa molecules that was immunoprecipitated from serologically reactive cell lines with each antibody. These antibodies also cross-reacted with two isoenzymes of alkaline phosphatase (strongly with the liver and bone, and moderately with the placental isoenzyme), but not with the intestinal form.
Structure of the antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella eimsbuttel
Authors: Fabio Jose L. Di , Perry Malcolm B. , Brisson Jean-Robert
Published: December 2009

The smooth lipopolysaccharide produced by Salmonella eimsbuttel, which had the O:6, O:7, and O:14 antigenic factors defined in the Kauffmann–White classification, was shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis, composition analysis, methylation, periodate oxidation, deamination, and 1H and 13C nuclear magnetic resonance studies to contain a high molecular weight O-chain polysaccharide composed of D-mannose (four parts), D-glucose (one part), and 2-acetamido-2-deoxy-D-glucose (one part). It was a branched polymer of a repeating hexasaccharide unit having the structure[Formula: see text]
Time-dependent alterations in the pathway of glucose incorporation into glycogen in primary cultures of rat hepatocytes following glucose starvation
Authors: Parniak M. A. , Kalant N.
Published: December 2009

Isolated rat hepatocytes maintained in primary culture were able to use glucose for glycogen synthesis by both direct and indirect mechanisms. Cells that had been isolated from fed animals and then cultured in the absence of glucose, but in the presence of gluconeogenic substrates such as pyruvate and amino acids, had decreased glycogen contents compared with similar cells that had been cultured in the presence of glucose. Upon reexposure to glucose, the glucose-starved cells showed time-dependent changes in the preferred pathway for the use of glucose for glycogen synthesis. These changes were noted either in the absence or presence of insulin; however, net accumulation of glycogen was observed only in the presence of the hormone.
Allosteric pyruvate kinase from Phycomyces blakesleeanus: physicochemical and regulatory properties
Authors: Busto Félix , Valle Pilar Del , Soler Joaquín
Published: December 2009

Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) was purified approximately 500-fold to a final specific activity of 25 U∙mg protein−1. The native enzyme has a molecular weight of 230 000. The enzyme appeared to be a tetramer with apparently identical subunits of 58 000 each. The enzyme from Phycomyces requires Mg2+ for activity, but not K+ or NH4. It showed a transition temperature at 36 °C. L-Alanine and ATP allosterically inhibited the enzyme by increasing the positive homotropic interactions for phosphoenolpyruvate and abolishing them for Mg2+ ions. Both effectors appeared to be competitive inhibitors with regard to ADP. Fructose 1,6-bisphosphate activates the Phycomyces pyruvate kinase allosterically by transforming the sigmoidal saturation curves to a hyperboolic form for phosphoenolpyruvate and Mg2+. Furthermore, fructose 1,6-bisphosphate relieved the inhibition caused by ATP and L-alanine. A lowering of the pH for reaction also activates the enzyme by abolishing the sigmoidal saturation curve for phosphoenolpyruvate, but produces substrate inhibition. The kinetic properties of Phycomyces pyruvate kinase are compatible to that of an allosteric K-type enzyme.
The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides
Authors: Rupar C. Anthony , Whitehall Jeffery D.
Published: December 2009

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze–thaw shock and membranes were isolated. The release of β-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.
Studies on protein phosphorylation using subcellular fractions from insulin-treated white adipose tissue of rats
Authors: Brownsey Roger W. , Dong Gordon W. , Lam Vivian , McGreer William
Published: December 2009

In these studies the incorporation of 32P into proteins within subcellular fractions, obtained from rat white adipose tissue upon incubation in the presence of [γ–32P] ATP, was investigated. A stable increase in the activity of protein serine(threonine) kinase in high-speed supernatant fractions was observed following treatment of intact tissue with insulin. Protein kinase activity associated with the plasma membrane fraction of cells was diminished in response to insulin, but the decrease was apparently insufficient to account for increases observed in corresponding supernatant fractions. A range of assay conditions was employed to characterize the insulin-stimulated protein serine(threonine) kinase in supernatant fractions. The insulin-stimulated protein serine(threonine) kinase displays properties that indicate it is distinct from a number of well-characterized protein kinases, including those regulated by cAMP, calcium ions (in the presence or absence of calmodulin or mixtures of phosphatidylserine–diacylglycerol), polyamines, or heparin. There were no apparent effects of insulin on incorporation of 32P into added casein or histones II-S or III-S. The protein serine(threonine) kinase activity (or activities) described here displays properties that also appear to differ from the properties of previously described insulin-stimulated activities able to catalyze the phosphorylation of the ribosomal protein S6. The differences in properties may, in part, be explained by the use of different cell types, but may also indicate that treatment of cells with insulin leads to activation of more than one protein serine(threonine) kinase.
Organelle origins and ribosomal RNA
Authors: Gray Michael W.
Published: December 2009

As the detailed molecular biology of organelle genomes has unfolded, there has been a general acceptance of the view that plastids and mitochondria are of endosymbiotic, eubacterial origin. Plastid genes are strikingly similar to their eubacterial (particularly cyanobacterial) counterparts in sequence, organization, and mode of expression, and such features strongly support the hypothesis that the plastid and its genome were derived in evolution from a blue-green alga-like endosymbiont. Mitochondria, on the other hand, are problematic: mitochondrial genes are organized and expressed in remarkably diverse ways in the different major groups of eukaryotes, and in no case are these features particularly characteristic of either bacterial or nuclear genomes. There is, however, clear evidence derived from gene sequence supporting the eubacterial ancestry of mitochondria, and some of the most compelling data have come from analyses of mitochondrial ribosomal RNA (rRNA). Plant mitochondrial rRNA genes diverge in sequence at a particularly slow rate, and these genes have proven to be especially supportive of the endosymbiont hypothesis, pointing to an origin of mitochondria from within the α subdivision of the purple bacteria. Ribosomal RNA sequences provide a basis for the construction of global phylogenetic trees that probe the evolutionary history of organelles, and that address the question of whether mitochondria and plastids are monophyletic or polyphyletic in origin. Such studies raise the possibility that the rRNA genes of plant mitochondria originated separately from the mitochondrial rRNA genes of other eukaryotes.
Taxol-induced reorganization of the microtubule system in murine splenic lymphocytes inhibits response to allogeneic cells but not to concanavalin A
Authors: Roy Chitra , Chaly Nathalie , Brown David L.
Published: December 2009

The exposure of mouse splenic lymphocytes to the microtubule assembly-promoting drug taxol (10 μM for 4 h) results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules, which extend from the centrosome. Lymphocytes pretreated with taxol for 4 h, or cultured in the continued presence of taxol, respond normally to the mitogen concanavalin A up to, and including, the stage of DNA replication. In contrast, the induction of DNA synthesis during the alloactivation of lymphocytes is inhibited when taxol is present in the mixed leukocyte culture. If the stimulators are pretreated with this drug, the mixed leukocyte reaction occurs normally, but pretreatment of the responders inhibits the proliferative response markedly. Microscopic observations of nuclear morphologies in these populations and autoradiography indicate that taxol inhibition occurs early in alloactivation, prior to DNA replication. The responding ability of taxol-treated lymphocytes is not restored to control levels by the addition of interleukin 2, leading to the suggestion that interleukin 2 receptors do not emerge or function normally in these cells. We conclude that the capacity to respond to allogeneic cells, but not to a mitogen, is dependent on the presence of the normal submembranous organization of the microtubule system.
Immobilized wheat germ agglutinin separates small oligosaccharides derived from poly-N-acetyllactosaminoglycans of embryonal carcinoma cells
Authors: Renkonen Ossi , Mäkinen Pekka , Hård Karl , Helin Jari , Penttilä Leena
Published: December 2009

Five pure oligosaccharides derived from poly-N-acetyllactosaminoglycans of teratocarcinoma cells were chromatographed on immobilized wheat germ agglutinin (WGA). Three of them, Galβ1-4GlcNAc, GlcNAcβ1-3Gal, and GlcNAcβ1-3Galβ1-4GlcNAc, revealed only weak binding, but GlcNAcβ1-6Galβ1-4GlcNAc was bound moderately and GlcNAcβ1-6Gal was bound quite strongly. The differences in the binding affinities were large enough to allow chromatographic separation of the five oligosaccharides into three distinct fractions in a small column of WGA–agarose.
The roles of epithelial–mesenchymal cell interactions in developmental processes
Authors: Sanders Esmond J.
Published: December 2009

This review surveys some recent trends in the study of the developmental interactions between epithelial and mesenchymal cells. The influence of such interactions on cell differentiation is considered with reference to kidney development, limb bud development, chondrogenesis and osteogenesis, and tooth development. Effects on epithelial morphogenesis are discussed, using salivary gland development as an example. The roles of humoral factors (hormones or growth factors) are considered, and the evidence for the participation of cell adhesion molecules is examined.
Regulation of SV40 early gene expression
Authors: Wildeman Alan G.
Published: December 2009

The early promoter of the simian virus 40 (SV40) has been used as a model eukaryotic promoter for the study of DN A sequence elements and cellular factors that are involved in transcriptional control and initiation. Site-directed mutagenesis and cell-free transcription systems have enabled the dissection of the functional domains within the 21 bp upstream sequence element and the 72 bp enhancer, and a number of protein factors that bind to various "motifs" within these domains have been identified. This article summarizes recent observations that have led to the conclusion that the SV40 promoter, and particularly, the enhancer region, is composed of multiple sequence elements. Some of these elements are present in cellular genes, and may exhibit tissue-specificity in their action. Furthermore, the proteins that are being identified (e.g., Sp1) may have binding sites within these elements that are sufficiently specific to ensure that only certain sets of genes will be selectively expressed.
Review: Modulation of chromatin structure by poly(ADP-ribosyl)ation
Authors: Murcia G. de , Huletsky A. , Poirier G. G.
Published: December 2009

Poly(ADP–ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosyl)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP–ribose) by poly(ADP–ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP–ribose) polymerase, which then synthesizes poly(ADP–ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.
Thyroid hormone induces synthesis and accumulation of tropomyosin and myosin heavy chain in limb buds of premetamorphic tadpoles
Authors: Dhanarajan Z. C. , Merrifield Peter A. , Atkinson Burr G.
Published: December 2009

Myosin heavy chain (MHC) and tropomyosin (Tm) have been isolated from limb muscles of the North American bullfrog, Rana catesbeiana, and injected into rabbits to raise monospecific antibodies. These antibodies were used to study the localization and synthesis of myosin heavy chain and tropomyosin in the limb buds of premetamorphic (stage VI–VII) tadpoles treated with triiodothyronine (T3) to induce metamorphosis. Indirect immunofluorescence localization detects the accumulation of both MHC and Tm in the developing thigh region within 24 h of T3 treatment. During the subsequent 48 h, the accumulation of these proteins is enhanced in the thigh and progresses from the thigh to the distal regions of the limb. Quantitative immunochemical determinations indicate that within 24 h of T3 treatment, synthesis of Tm and MHC are increased 23-fold and 6-fold, respectively. Following 5 days of T3 treatment, the synthetic rates of Tm and MHC are 266 and 70 times the control values, respectively. Both methods suggest that Tm is synthesized and accumulated at a greater rate than myosin heavy chain. These observations suggest that T3 promotes the differentiation of muscle in the limb buds of premetamorphic tadpoles and that limb development promoted by T3 in tadpoles is similar to that described during the embryonic development of higher vertebrates.
Purification and characterization of a murine cellular retinoic acid-binding protein
Authors: Bailey John Stuart , Siu Chi-Hung
Published: December 2009

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE 9–4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14 600 in SDS – polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins, However, it is immunologically distinct from the cellular retinol-binding protein.
The release of radioactive arachidonate from lipids of red blood cell membranes: properties of membrane and serum enzymatic activities
Authors: Baker R. Roy , Loh Zou Dao
Published: December 2009

The release of [3H]arachidonate from the phospholipids of red blood cell membranes of rats has been studied. Membranes of red cells isolated using acid–citrate–dextrose and differential centrifugation showed an endogenous release of arachidonate at pH 7.4 in the presence of CaCl2. Membranes from red cells isolated using heparin and Percoll gradient centrifugation are better substrates for serum-mediated release of arachidonate. These experiments and results with inhibitors suggest that red blood cell and serum phospholipase A2 activities are responsible for this catabolism that may provide arachidonate for subsequent biosynthesis of eicosanoids.
Binding specificity and reactivity studies on a broad-specificity β-glycosidase from porcine kidney
Authors: Huber R. E. , Brockbank R. L.
Published: December 2009

A broad-specificity β-glycosidase from porcine kidney was purified to homogeneity. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis showed that it had a monomeric molecular weight of 55 000–60 000. Gel filtration showed a native molecular weight of about 115 000. These data imply that the native enzyme is a dimer. The enzyme can catalyze the hydrolysis of β bonds between glycosides and 4-methylumbelliferone or nitrophenol yielding D-fucopyranose, D-galactopyranose, D-glucopyranose, D-xylopyranose, and D-mannopyranose and of α bonds to yield L-arabinopyranose. This is the first study that shows a mammalian broad-specificity cytosolic β-glycosidase carrying out a reaction with a β-D-mannopyranoside. The nature of the broad specificity was studied with inhibitors. Similar inhibitor constants were found regardless of whether the substrate was a β-D-glucopyranoside or a β-D-galactopyranoside, so the enzyme probably has only one binding site with a broad specificity. The enzyme prefers to bind compounds with an axial hydroxyl at the 2 position and an equatorial hydroxyl at the 4 position; the 3 position does not affect binding significantly. The hydroxyl at the 6 position affects binding, but binding at that position depends on the configurations at the 2 and 4 positions. Thus, there must be some interactions between these three positions (2, 4, and 6). Lactones are also good inhibitors and this may relate to strain effects.
Screening of monoclonal antibodies for hCG for affinity and specificity
Authors: Leblond Lorraine , Lemieux Édith , Patel Praful , Crine Philippe
Published: December 2009

Using human chorionic gonadotropin (hCG) as a model polypeptide, we have developed a strategy that allows the direct screening of supernatant fluids from hybridomas for the presence of monoclonal antibodies of high affinity and predefined specificities. The assay evaluates the competition between 125I-labeled and unlabeled homologous or heterologous antigens in a solid-phase two-site immunoradiometric assay. This assay is fast and accurate, and is of general use provided the antigen of interest can be purified in nanomolar quantities. This strategy led to the isolation of nine new monoclonal antibodies for hCG, two of which could be used for elaborating a sensitive two-site immunoradiometric assay for this hormone.
Characterization of high-density lipoprotein binding to rat adipocytes and adipocyte plasma membranes
Authors: Zsigmond Eva , Fong Bessie , Angel Aubie
Published: December 2009

The interaction of high-density lipoproteins (HDL) with adipocytes is important in the regulation of cellular cholesterol flux. To study the mechanisms of HDL binding and cellular processing, we incubated adipocytes isolated from epididymal and perirenal adipose tissue of male Wistar rats (300 g) with HDL1 (1.07–1.10 g/mL) and HDL2 (1.10–1.14 g/mL) fractions separated from rat plasma by gradient ultracentrifugation. Freshly isolated adipocytes were incubated with 125I-labeled HDL for 2 h at 37 °C to determine cell-associated uptake and degradation. Adipocytes from both fat regions showed significant cell-associated HDL1 and HDL2 uptake and very high medium degradation (2- to 6-fold higher than uptake). To assess 125I-labeled HDL binding independent of cellular metabolism, we purified adipocyte plasma membranes from isolated adipocytes and used them in binding assays. Binding of HDL1 and HDL2 in the membrane system was 85–95% specific, sensitive to high NaCl concentrations, and abolished by pronase treatment. In contrast to HDL2 binding, the maximum HDL1 binding to perirenal plasma membranes was significantly higher than its binding to epididymal membranes (7.2 ± 1.3 vs. 4.4 ± 0.2 μg/mg, n = 6, p < 0.05). This increment in HDL1 binding to perirenal membranes represented an EDTA- sensitive, calcium-dependent component. These results indicate that HDL binding to adipocyte plasma membranes depends on both adipose tissue region and HDL subtype. The membrane binding characteristics, taken together with the cellular uptake results, suggest that adipocytes bind and metabolize HDL and that this interaction may involve a protein receptor.
Aortic stenosis produces hypertrophy of the rat heart without causing an accumulation of N1-acetylspermidine
Authors: Mezl Vasek A. , Villemure Christiane , Garber Pascale M. , Rakusan Karel
Published: December 2009

Isoprenaline treatment causes cardiac hypertrophy and an accumulation of N1-acetylspermidine in the rat heart. To determine whether the cardiac hypertrophy is the cause of the increase in N1-acetylspermidine, we produced cardiac hypertrophy by constriction of the aorta and analyzed polyamines in the hearts of these rats 1, 3, and 10 days after the aortic constriction. Our results show that compared to sham-operated animals, this treatment caused a 60% increase in putrescine and a 30% increase in spermidine by day 10, but not the expected increase in N1-acetylspermidine. We conclude that N1-acetylspermidine is not induced by a cardiac overload.
Effect of 3-deazaadenosine on methionine metabolism in isolated perfused livers
Authors: Duerre John A.
Published: December 2009

The effect of the purine analog 3-deazaadenosine (dzAdo) on the metabolism of sulfur-containing compounds was examined in hepatocytes. The uptake of exogenous methionine by the liver was not affected by the addition of dzAdo to the perfusate, while the intracellular concentrations of S-adenosyl-L-methionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) continued to increase as long as exogenous methionine was available. In addition, large amounts of 3-deazaadenosyl-L-homocysteine (dzAdoHcy) accumulated in the cell. The specific radioactivity of the carbon chain of dzAdoHcy was the same as that of AdoMet and AdoHcy. Consequently, an equivalent amount of homocysteine (Hey) must have been generated via hydrolysis of AdoHcy. Free Hey could not be detected either in the tissue or perfusate when dzAdo was present, while Hey was excreted into the perfusate by control livers. Consequently, the AdoHcy and DzAdoHcy that accumulate in the cell not only function as inhibitors of methylation reactions, but serve as a trap for Hey. This could result in methionine starvation and hence, inhibition of protein synthesis.
Structure of the O-chain of the lipopolysaccharide of Pasteurella haemolytica serotype T3
Authors: Leitch Robert A. , Richards James C.
Published: December 2009

Cell-wall lipopolysaccharide isolated from Pasteurella haemolytica serotype T3 using the phenol–water extraction procedure was shown to be an S type lipopolysaccharide by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Hydrolysis with mild acid afforded a lipid-free, antigenic O-chain polysaccharide. On the basis of one- and two-dimensional 1H and 13C nuclear magnetic resonance studies, in conjunction with microanalytical chemical methods, the O-polysaccharide was determined to be a linear polymer of a disaccharide repeating unit having the structure[Formula: see text]
Changes in phospholipid metabolism dependent on calcium-regulated myoblast fusion
Authors: Sauro Victor S. , Brown Gregory A. , Hamilton Michael R. , Strickland Craig K. , Strickland Kenneth P.
Published: December 2009

Fusion-competent myoblasts can be prevented from fusing (differentiating) by reducing medium calcium concentrations from 1.65 mM to less than 50 μM. Fusion is completely retarded after 24 h but is noticeable after 48 h and significant after 72 h in low-calcium medium. After 24 h in low-calcium medium, a rapid, synchronous fusion can be initiated by return to normal (high-calcium) medium. Calcium content increases over threefold during myoblast differentiation and closely parallels the fusion process. Phospholipid content is also dependent upon the state of differentiation. Myotubes (fused myoblasts) have an almost twofold greater content of lipid phosphate per milligram of protein compared with that of myoblasts; this increase is localized to increased contents of phosphatidylcholine and pooled phosphatidylinositol – phosphatidylserine. Phospholipid synthesis (32Pi incorporation) is markedly stimulated four- to five-fold when myoblasts grown in low-calcium medium are switched to normal medium. These significant increases are observed in all the major phospholipids studied, predominantly in phosphatidylcholine and pooled phosphatidylinositol – phosphatidylserine, and most noticeably in phosphatidylinositol 4,5-bisphosphate. Furthermore, we show that phosphatidylinositol 4,5-bisphosphate prelabelled with myo-[2-3H]inositol is rapidly degraded after switching from low-calcium medium to normal medium. These changes are not observed in myotubes treated similarly, which suggests that the changes in phospholipid metabolism may be fusion related. These results support a proposal by another author, which suggests that phosphatidylinositol 4,5-bisphosphate breakdown may play an important regulating role in myoblast differentiation.
Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes
Authors: Brockhausen Inka , Carver Jeremy P. , Schachter Harry
Published: December 2009

Glycoproteins isolated from hen oviduct contain highly branched asparagine-linked oligosaccharides (N-glycans). Six N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V, and VI) are involved in initiating the synthesis of these branches, as indicated below:[Formula: see text]where R is GlcNAcβ1—4(+/−Fucα1—6)GlcNAcAsn-X. HPLC has been used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct. Oligosaccharides with free reducing GlcNAc termini were prepared from various glycoproteins by hydrazinolysis–re-N-acetylation and used as GlcNAc-T substrates and HPLC standards. Enzyme assay components were separated on AG1 × 8, followed by HPLC on amine-bonded silica columns eluted with acetonitrile–water mixtures. Absorbance at 195 nm and radioactivity of eluted compounds were monitored. Substrates and products were identified by comparison of their retention times with those of oligosaccharides with known structures. Enzyme assay by HPLC is more rapid and convenient than previous GlcNAc-T assays using lectin columns or electrophoresis. Since some substrates yielded multiple products, these could be used to assay more than one GlcNAc-T in the same incubation. GlcNAc-T VI was shown to act on both bisected and nonbisected GlcNAc-terminating tetraantennary oligosaccharide substrates; GlcNAc-T II, IV, and V acted poorly or not at all on bisected substrates. GlcNAc-T V was the only enzyme among the six transferases studied that could be assayed in the absence of Mn2+.
Evidence that the acute unmasking of GDP-binding sites in brown adipose tissue mitochondria is not dependent on mitochondrial swelling
Authors: Milner Rachel E. , Trayhurn Paul
Published: December 2009

The role of mitochondrial swelling in the unmasking of GDP-binding sites on brown adipose tissue mitochondria has been examined in mice. Acute cold exposure (6 °C for 1 h) led to increases in GDP binding without changes in the concentration of uncoupling protein, indicating that an unmasking of binding sites had occurred. Measurements of mitochondrial matrix volume suggested that an acute unmasking of GDP-binding sites took place without swelling of the mitochondria. In addition, the induction of a rapid preswelling of the mitochondria by incubation in KCl in the presence of valinomycin did not affect the cold-induced unmasking of GDP-binding sites. It is concluded that the acute unmasking of GDP-binding sites on uncoupling protein in brown adipose tissue is not due simply to mitochondrial swelling.
Differential effect of inflammation and dexamethasone on dolichol and dolichol phosphate synthesis
Authors: Sarkar Mohan , Mookerjea Sailen
Published: December 2009

Inflammation and glucocorticoids stimulate hepatic glycoprotein synthesis, resulting in an increased secretion of serum glycoproteins. We now present evidence that the synthesis of dolichol and dolichol phosphate from mevalonate is increased in hepatocytes from inflamed rats. Also, in inflamed rats, the levels of dolichol and dolichol phosphate are increased in liver homogenates and microsomes. Dexamethasone treatment of the cells, however, does not increase the synthesis of dolichol and dolichol phosphate from mevalonate. The results suggest that the inflammation-induced dolichol-linked saccharide and glycoprotein synthesis is possibly mediated through an increase in the level of dolichol and dolichol phosphate in the liver. Since dexamethasone treatment does not increase the synthesis of dolichol and dolichol phosphate, its action on glycoprotein synthesis appears to be different and to affect the induction of enzymes in mannosyl phosphoryl dolichol- and dolichol-linked oligosaccharide synthesis.
The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells
Authors: Forster-Gibson Cynthia J. , Fei Yu Xiao , Dufresne Michael J.
Published: December 2009

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p < 0.005), two had approximately the same, and two had significantly higher (p < 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 ± 0.09 for clone 1 (Hs-1) and 0.94 ± 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p < 0.005), two had approximately the same, and one had significantly higher levels (p < 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).
Lysosphingolipids and mitochondrial function. II. Deleterious effects of sphingosylphosphorylcholine
Authors: Strasberg Paula M. , Callahan John W.
Published: December 2009

Psychosine, sphingosylphosphorylcholine (52–104 μM), and other glycosphingolipids stimulate mitochondrial respiration (up to 500%) and inhibit oxidative phosphorylation to varying degrees. Above 104 μM these functions as well as uptake of Ca2+ are prevented. At 104 μM sphingosylphosphorylcholine inhibits the mitochondrial ATPase reaction in submitochondrial particles by 48%. Both sphingosylphosphorylcholine and psychosine enhance the active phosphate-dependent swelling of mitochondria. Passive swelling occurs in the presence of rotenone (when swelling does not normally occur) and under hypotonic conditions. A direct interaction of sphingosylphosphorylcholine with membranes is demonstrated by a discharge of the proton gradient across mitochondrial membranes, hemolysis of red blood cells, and binding to inner and outer mitochondrial membranes. Thus lysosphingolipids bind strongly to mitochondrial membranes and markedly alter mitochondrial function. This alteration would affect the ATP levels, thereby altering a wide range of ATP-dependent cellular functions. These results offer a partial explanation for the pathogenesis of representative lysosomal storage diseases.
Metabolism of nitrogen fixed by nodules of alfalfa (Medicago sativa L.): II. Asparagine synthesis
Authors: Ta T.-C. , Macdowall F. D. H. , Faris M. A. , Joy K. W.
Published: December 2009

Asparagine is a major nitrogen transport compound in many legumes including alfalfa. In vivo experiments with nodulated alfalfa roots exposed to labeled 15N2 and 14CO2 revealed that asparagine is synthesized at a rate of 150 nmol∙gFW nodule−1∙min−1. Nodule segments incorporate [U-14C, 15N]aspartate into asparagine, and the 14C/15N (amino) ratio of asparagine is very similar to that of aspartate. The results show that asparagine is synthesized mainly by the amidation of aspartate utilizing the amide group of glutamine; however, there is evidence that ammonia can also contribute directly to asparagine synthesis. The in vitro activity of asparagine synthetase in nodule extracts was detected at a maximum rate of 44 nmol∙gFW−1∙min−1 through the inclusion of sulfhydryl reagents, EGTA, and glycerol in the extract buffer. The enzyme requires aspartate, glutamine (or ammonia), and ATP as substrates. Activity increases during nodule development, reaching a maximum value 17 days after inoculation. The measured rates for asparagine synthesis and enzyme activity are much higher than previously reported.
The lipid composition of plasma membrane from goat mammary gland
Authors: Sharma Arun , Dahiya Rajvir
Published: December 2009

Experiments were conducted to examine and characterize the lipid composition of the plasma membrane from the lactating goat mammary gland. The plasma membranes were purified by discontinuous sucrose density centrifugation. Lipids were extracted from these membranes and analyzed by thin-layer and gas–liquid chromatography. The results of these studies demonstrate that (i) the principal phospholipids of mammary-gland plasma membranes are phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; (ii) the principal neutral lipids are triacylglyceride and cholesterol ester; (iii) the major glycolipids are globotetraosylceramide and globotriaosylceramide; and (iv) the major fatty acids are oleic (18:1), palmitic (16:0), stearic (18:0), and myristic (14:0) acids.
Rapid purification of ferritin from lysates of red blood cells using proteinase-K
Authors: Atkinson Burr G. , Dean Rob L. , Tomlinson Jeff , Blaker Timothy W.
Published: December 2009

A simple, rapid, and novel procedure for purifying ferritin from the postnuclear supernatant of red blood cell lysates is described. This report establishes the resistance of commercially available holo- and apo-ferritins to proteinase-K digestion, and documents how the use of this enzyme, in conjunction with the well-documented resistance of ferritins to heat denaturation (75–80 °C for 10 min), makes it possible to obtain high yields (> 90%) of pure, undegraded ferritin from the postnuclear supernatant of hypotonically or Triton X-100 lysed red blood cells. The resultant purified ferritin contains the same amount of iron as ferritin not treated with proteinase-K and, as judged by one- and two-dimensional gel electrophoresis and electron microscopy, consists of intact ferritin with a subunit isoform composition identical in molecular mass and isoelectric points to that obtained from ferritin prepared in the absence of this enzyme.Key words: ferritin, proteinase-K, ferritin purification, erythrocytes.
Structural analysis of the three lipopolysaccharides produced by Salmonella madelia (1,6,14,25)
Authors: Fabio José L. Di , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

Salmonella madelia reported to express the O-antigenic factors 1, 6, 14, and 25, defined in the Kauffmann–White classification system, was found to produce three different homogeneous lipopolysaccharides, which differed in having three structurally distinct O-polysaccharide components. The O-polysaccharide fraction obtained by mild acetic acid hydrolysis of the S. madelia lipopolysaccharide was analyzed by chemical composition, nitrous acid deamination, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance methods and was demonstrated to be composed of three polysaccharides, PS(I), PS(II), and PS(III), which had the structures of repeating oligosaccharide units:[Formula: see text]Key words: Salmonella madelia, lipopolysaccharide, structure, analysis, nuclear magnetic resonance.
Characterization of the heat-shock response in spinach (Spinacia oleracea L.)
Authors: Somers Daryl J. , Cummins W. Raymond , Filion W. Gary
Published: December 2009

Spinach (Spinacia oleracea L. cultivar Longstanding Bloomsdale) grown at 20 °C was subjected to a range of rapid thermal shifts as high as 42 °C. There was a decrease in the level of protein synthesis following heat-shock treatments above 34 °C as indicated by the level of incorporation of L-[35S]methionine. In vivo labelled polypeptides and in vitro translation products of RNA isolated from leaf tissue and analyzed using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography, indicated that the temperature of induction of all 15 heat-shock proteins in the 20 °C grown plants was 36 °C. In addition, heat-shock RNA was coordinately expressed and the translation of heat-shock proteins was noncoordinate with respect to temperature. Treatment with cycloheximide and with chloramphenicol demonstrated that heat-shock protein synthesis in spinach was restricted to cytosolic ribosomes. Synthesis of some low molecular weight heat-shock proteins were insensitive to actinomycin D, suggesting greater stability of these heat-shock RNAs. The heat-shock polypeptide profile of plants grown at 10 °C was similar to that of plants grown at 20 °C, with 14 heat-shock proteins being induced at 36 °C. The growth temperature did not influence the final array of heat-shock proteins synthesized nor alter the temperature of induction of the heat-shock response.Key words: heat-shock response, heat-shock proteins, Spinacia oleracea.
Developmental regulation of cell-surface glycoproteins in clonal cultures of human skeletal muscle satellite cells
Authors: Champaneria Shashikant , Holland Paul C. , Karpati George , Guérin Claude
Published: December 2009

Pure populations of myogenic cells were obtained by cloning satellite cells from human skeletal muscle biopsies. Cell-surface glycoproteins at various stages of myogenesis were analysed by one- and two-dimensional gel electrophoresis. A total of 14 distinct proteins were detectable at the cell surface, on the basis of their susceptibility to desialation by exogenous neuraminidase or their iodination by exogenous lactoperoxidase. Reproducible changes in lectin binding or iodination of eight of these proteins occurred during myogenesis. Only two of the developmentally regulated proteins were components of the detergent-insoluble extracellular matrix fraction. Developmental regulation of these two proteins was unaffected by growth of cultures in 5-bromo-2′-deoxyuridine to inhibit myogenesis. In contrast, developmental regulation of the other cell-surface proteins was inhibited by growth in 5-bromo-2′-deoxyuridine, suggesting that changes in these proteins are tightly coupled to satellite cell differentiation. These studies represent the first systematic analysis of the surface proteins of pure, clonally derived, primary cultures of normal myogenic cells.Key words: satellite cells, myogenesis, myoblast, glycoproteins, cell surface.
Examination of heat shock gene expression in nerve cord isolated from heat-stressed American cockroach, Periplaneta americana
Authors: Ruder G. K. , Ovsenek N. , Heikkila J. J. , Downer R. G. H.
Published: December 2009

Exposure of adult female cockroaches to temperatures ranging from 37 to 42 °C induced the synthesis of a set of heat shock proteins (HSPs) in isolated nerve cord. The major HSPs have molecular masses of 83 000, 70 000, 68 000, and 28 000 daltons. Enhanced synthesis of these HSPs was detectable after 15 min of heat shock and approached maximum levels after 2 h. In cockroaches exposed to 42 °C for 1 h and then returned to 22 °C, a normal pattern of protein synthesis was established within 3–5 h. Northern hybridization analysis using Drosophila HSP 70 and HSP 83 genomic clones revealed the accumulation of 2.6 and 3.1 kilobases mRNA, respectively, in total RNA derived from heat-shocked tissue. A rapid decay of these mRNAs was observed in cockroach nerve cord during recovery at 22 °C following a 1-h heat shock at 42 °C. The decrease in HSP synthesis and HSP mRNA levels during recovery may be due to a decrease in mRNA stability as well as transcription.Key words: heat shock proteins, Periplanata americana, nerve cord, gene expression, mRNA.