bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose oxidation by adherent and mechanically detached cultured cells
Authors: Honda B. D. , Glanville N. T.
Published: December 2009

This study examined the effect of mechanical detachment from the growth surface on energy metabolism of cultured cells. Oxidation of [1-14C]glucose measured by production of 14CO2 by adherent neuroblastoma (123 ± 5 nmol/mg protein per minute), glioma (128 ± 10 nmol/mg protein per minute), and fibroblast (137 ± 5 nmol/mg protein per minute) cultures was similar. Removing cells from the culture flask by scraping reduced glucose oxidation by 62, 30, and 82% in neuroblastoma, glioma, and fibroblast cultures, respectively. Transferring cells from a culture flask to a test tube, to control for diffusional surface area, did not further reduce glucose oxidation. Detaching cells from the growth surface destroyed the extensive process formation and disrupted the normal spatial organization on the culture plate. These results indicate that it is essential to maintain these aspects of cellular architecture when evaluating metabolic properties of cultured cells.Key words: glucose oxidation, neuroblastoma, glioma, fibroblasts.
Inability of insulin and insulinlike growth factor-1 to stimulate sugar or amino acid transport and thymidine incorporation in cultured myeloma cells
Authors: Leckett Blaine , Germinario Ralph J.
Published: December 2009

NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 × 10−10 M) or IGF-1 (i.e., 1.5 × 10−10 M), NS-1 cells specifically bind 0.2 ± 0.06 fmol insulin per 106 cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 ± 0.01 fmol/106 cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 ± 390 (n = 3). Insulin was employed at various concentrations (6.7–667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4–143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.Key words: cultured myeloma cells, insulin and IGF-1 binding and action, sugar transport, amino acid transport, DNA synthesis.
Differential scanning calorimetry and X-ray diffraction studies of a series of synthetic β-D-galactosyl diacylglycerols
Authors: Mannock D. A. , McElhaney R. N.
Published: December 2009

We have investigated the physical properties of a homologous series of synthetic, saturated 1,2-di-O-acyl-3-O-(β-D-galactopyranosyl)-sn-glycerols using calorimetry and X-ray diffraction. Unannealed aqueous dispersions of these compounds exhibit a lower temperature, moderately energetic, chain-melting (Lβ/Lα phase transition and a higher temperature, weakly energetic, bilayer/nonbilayer phase transition. On annealing below the Lβ/Lα phase transition, the Lβ phase converts to an LC phase, which may undergo a highly energetic LC/Lα or LC/HII phase transition at very high temperatures on reheating. The temperatures of these phase transitions are higher than those seen in the corresponding α- and β-D-glucosyl diacylglycerols. However, the Lβ/Lα and bilayer/nonbilayer phase transition temperatures of the β-D-galactosyl diacylglycerols are lower than those of the corresponding diacyl phosphatidylethanolamines. These observations are discussed in terms of the hydration and hydrogen bonding properties of their respective headgroups.Key words: differential scanning calorimetry, low-angle x-ray diffraction, glycolipids, galactolipids, lipid phase behaviour.
Stabilization of the structure of horse plasma vitamin D binding protein by disulfide bonds
Authors: Robinson Robert C. , Burtnick Leslie D.
Published: December 2009

Vitamin D binding protein (DBP) was isolated from horse plasma in a four-step procedure that involved Affi-Gel Blue affinity chromatography, gel filtration, hydroxylapatite chromatography, and anion exchange high-pressure liquid chromatography. The yield of DBP from 80 mL of plasma was 6–7 mg. Horse plasma DBP closely resembles other plasma DBPs, being a tryptophan-free protein of Mr 53 000. It is able to bind to and block the polymerization of monomeric actin. The secondary structure of DBP was calculated from circular dichroism measurements to be 39% α-helix, 42% β-sheet, and 19% random coil. Circular dichroism and fluorescence studies revealed that the disulfide bonds of DBP contribute substantial structural stabilization to the molecule with respect to thermal denaturation. The thermal stability of DBP can be used to advantage. Incorporation of a brief treatment at 70 °C into the preparative scheme enables omission of one chromatographic step, without detectable alteration of the purified product.Key words: vitamin D binding protein, circular dichroism, disulfide bonds, thermal denaturation, fluorescence.
Intermediate filaments in rat ovarian surface epithelial cells: changes with neoplastic progression in culture
Authors: Hornby Ann E. , Pan Jie , Auersperg Nelly
Published: December 2009

Interrelationships between neoplastic progression and the expression of intermediate filaments were examined in primary cultures, immortal lines, and Kirsten murine sarcoma virus (KiMSV) transformed lines of rat ovarian surface epithelial (ROSE) cells. Immunofluorescence microscopy revealed abundant keratin filaments in all cells of primary cultures. In immortal, nontumorigenic lines, keratin filaments were detected in fewer cells, in smaller numbers, and in microscopically altered forms. The percentage of keratin-positive cells ranged from 4 to 54%. Its expression was inversely proportional to cell density. Keratin expression was similar in the two immortal lines, although one had retained a monolayered epithelial growth pattern resembling primary cultures, while in the other the growth pattern of the cells was more atypical. The two KiMSV-transformed lines were previously shown to produce tumors in vivo that resemble human ovarian endometrioid stromal sarcomas. In spite of this histologic appearance, the proportion of keratin-positive cells in these cells was increased over the immortal lines. Keratin expression was unrelated to cell density, and keratin in most virally transformed cells was limited to few, fine filaments. In thymidine-labelled immortal and virus-transformed cultures stained for keratin, no correlation was found between keratin expression and proliferative activity. The keratin profiles of primary and immortal cultures were identical on Western blots, with subtypes ranging from 52 to 66 kDa. The two virally transformed lines lacked some of the subtypes. Vimentin networks were faint or absent in primary cultures. In the immortal and the virus-transformed lines, neoplastic progression was associated with increasing vimentin expression but with no changes in filament morphology and distribution. The results show that the abnormalities in intermediate filament expression that accompany immortalization do not preclude the retention of a normal epithelial morphology and growth pattern in this cell type. Furthermore, the number of intermediate filaments and their intracellular distribution appear to be altered at an earlier stage in neoplastic progression than those mechanisms that select for specific keratin subtypes, or those that respond to regulation by cell density. Finally, the presence of keratin in the KiMSV-transformed lines examined in this study supports the hypothesis that human ovarian stromal sarcomas can arise in the OSE.Key words: neoplastic transformation, keratin, vimentin, cytoskeleton, ras oncogene, ovary, tissue culture.
Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity
Authors: Ghosh S. S. , Franson Richard C.
Published: December 2009

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0–8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 μM) or propanolol (IC50 = 180 μM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.Key words: Escherichia coli, substrate, phospholipase D, Streptomyces chromofuscus, sodium fluoride, propranolol.
Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+
Authors: Sage E. Helene
Published: December 2009

SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca2+-binding protein that inhibits the spreading of newly plated cells and elicits a rounded morphology in spread cells. In this study, I investigated whether the rounding effect of SPARC depends on the ability of the protein to chelate Ca2+ at the cell surface. Bovine aortic endothelial cells were plated in the presence of different concentrations of SPARC and Ca2+; control experiments were performed with 1 mM EGTA and with Mg2+. Quantitative estimates of cell rounding were calculated according to a rounding index. SPARC, at concentrations between 0.15 and 0.58 μM, elicited rounding (or prevented spreading) of cells cultured for 16–38 h in 0.5–2.0 mM Ca2+. Addition of 0.5–2.0 mM Mg2+ to cells previously rounded in the presence of SPARC did not abrogate the effect of SPARC. When the levels of extracellular Ca2+ were adjusted with 1 mM EGTA to maximum values ranging from 7.1 to 320 μM, cells displayed a rounded morphology in the presence of exogenous SPARC. Although the rounding induced by 1 mM EGTA was essentially reversed by the inclusion of 2 mM Ca2+, cultures containing these reagents together with SPARC maintained the rounded phenotype. These results do not support a mechanism that involves the abstraction of Ca2+ from proteins at the cell surface or the provision of Ca2+ from native extracellular SPARC to cells. Therefore, SPARC does not appear to act as a local chelator of extracellular Ca2+ and Mg2+ and presumably exerts its function as a modulator of cell shape via a different pathway.Key words: SPARC, osteonectin, endothelial cells, cell shape.
Fatty acid uptake in Candida tropicalis: induction of a saturable process
Authors: Trigatti Bernardo L. , Baker Andrew D. , Rajaratnam Krishan , Rachubinski Richard A. , Gerber Gerhard E.
Published: December 2009

The rates of oleate uptake by Candida tropicalis cells grown on a high oleate concentration (3.5 mM oleate in the presence of 0.50% Brij 58) were higher than those observed in cells grown on glucose; however, oleate uptake was not saturable with substrate concentration. Cells grown at a low oleate concentration (1.0 mM oleate and 2.5% Brij 58) grew to a lower density and at a slightly slower rate; these cells were found to take up oleate at a rate 43-fold higher than cells grown on high oleate concentration. Furthermore, oleate uptake by the cells grown in low oleate medium was a saturable process with Kt and Vmax values of 56 μM and 15 nmol/(min∙mg cell protein), respectively. The growth of C. tropicalis under low fatty acid concentration thus clearly results in the induction of a saturable process for its uptake. The total level of acyl-CoA synthetase activity in cells grown on the low oleate concentrations was only twofold higher than in high oleate or glucose grown cells; the level of this enzyme thus does not account for the saturable process and suggests that either the enzyme is regulated in vivo or else a hitherto unidentified enzyme is induced by growth in low concentrations of oleate.Key words: acyl-CoA synthetase, Candida tropicalis, induction, oleate, uptake.
Endogenous substrates of rat heart protein kinase C type I, II, and III isozymic forms in cardiac sarcolemma
Authors: Qu Yi , Torchia Joseph , Phan Thanh Duc , Wu Po Hsiung , Sen Amar Kumar
Published: December 2009

The endogenous substrate proteins of rat cardiac protein kinase C type I, II, and III isozymic forms were studied in rat cardiac sarcolemma. The 19-, 21-, 29-, 35-, and 95-kDa proteins were phosphorylated by both types II and III, but not type I. The extent of phosphorylation by individual protein kinase C isozymic forms was additive and equal to the extent of phosphorylation observed when a mixture of isozymic forms was employed. The extent of phosphorylation of the 21-kDa protein by type III was much higher than that by type II. These results suggest that the protein kinase C isozymes have preferences for specific endogenous substrate proteins. The phosphorylation of these endogenous substrate proteins by protein kinase C isozymes probably plays a role in cardiac cell functions.Key words: cardiac sarcolemma, protein kinase C isozymes, phosphorylation, substrate proteins.
Enzymatic in vitro synthesis of radiolabeled pentasaccharides GlcNAcβ1-3(Galβ1-4GlcNAcβl-6)Galβ1-4GlcNAc/Glc and the isomeric Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc/Glc
Authors: Renkonen Ossi , Leppänen Anne , Niemelä Ritva , Vilkman Anja , Helin Jari , Penttilä Leena , Maaheimo Hannu , Seppo Antti , Suopanki Jaana
Published: December 2009

Four radiolabeled pentasaccharides, GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc, Galβ1-4GlcNAcβ1-3-(GlcNAcβ1-6)Galβ1-4GlcNAc, GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc, and Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)-Galβ1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic β1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).Key words: In vitro synthesis, branched pentasaccharides, backbones of oligo(N-acetyllactosaminoglycans).
Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase from rat liver
Authors: Brockhausen Inka , Möller Gabriele , Pollex-Krüger Annette , Rutz Volker , Paulsen Hans , Matta Khushi L.
Published: December 2009

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase (β3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, β4-galactosyltransferase, β-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the β3-Gal-T. β3-Gal-T activity is sensitive to changes in the R-group of the GalNAcα-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAcα-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished β3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAcα-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. Derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAcα-benzyl, which inhibited Gal incorporation into GalNAcα-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAcβ6[Galβ3]GalNAcα-) is formed from core 1 (Galβ3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.Key words: β3-Gal-transferase, mucin synthesis, O-glycan core 1, enzyme specificity, enzyme inhibition.
Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells
Authors: Mello Maria Luiza S. , Vidal Benedicto de C. , Golembieski William , Russo Jose
Published: December 2009

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen–DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.Key words: cell transformation, 4-nitroquinoline 1-oxide, NIH/3T3 cell assay, human breast epithelial cells, image analysis.
Expression of c-fos and c-myc genes in uterus and nonreproductive organs during the implantation period in the rat
Authors: Verneaux V. , Jouvenot M. , Compagnone P. , Propper A. Y. , Adessi G. L.
Published: December 2009

The c-fos and c-myc mRNA levels were investigated in the uterus and nonreproductive organs of rats during the implantation period (from day 5 to 7 of pregnancy). They were determined by densitometric analysis of slot blots and the mean values (n = 4) at a defined age of pregnancy were compared with those observed in nonpregnant control rats (NP group). No significant variations of c-fos level were observed in the liver and brain of pregnant rats. In the uterus, the c-fos level decreased, reached a minimum on day 6 (4-fold decrease compared with the NP group), and then increased. The c-fos level significantly increased in the kidney (2- and 2.5-fold on days 6 and 7), and in the lung (2.5-fold on day 7). The c-myc level did not significantly vary in the uterus, liver, and lung. It significantly increased in the kidney (3-fold on day 7) and in the brain (4.1-, 3.8-, and 6-fold, on days 5, 6, and 7). The changes in c-fos and (or) c-myc gene expression in the uterus and nonreproductive organs could be due to sexual steroids and (or) systemic factors from uterine cells or blastocysts.Key words: pregnancy, implantation, uterus, nonreproductive organs, c-fos, c-myc.
Repression of a G0-associated 65-kilodalton protein in actively proliferating and SV40-transformed mouse kidney cells
Authors: Rose Timothy M. , Tremblay Sandra , Khandjian Edward W.
Published: December 2009

The pattern of [35S]methionine-labeled proteins from primary cultures of mouse kidney epithelial cells arrested in G0 phase was analyzed by two-dimensional gel electrophoresis and compared with that observed from cultures of actively proliferating and SV40-transformed mouse kidney cells. A major polypeptide (p65) migrating with a molecular mass of 65 000 daltons and a pI of 5.8 was detected in quiescent cultures of cells which had exhausted their finite division potential. Under the experimental conditions used, these cells had lost sensitivity to growth factors and were irreversibly blocked in G0 phase of the cell cycle. In cultures of actively proliferating mouse kidney cells, the expression of p65 was not observed until just prior to arrest. Moreover, proliferating cultures of immortalized mouse kidney cells that had been reactivated from their quiescent state by infection with SV40 did not express p65. Subcellular localization studies suggest that p65 is associated with the crude nuclear fraction. In addition, p65 is glycosylated and binds the lectin concanavalin A. Pulse–chase experiments demonstrated that p65 was short lived with an estimated half life of 10 min. Thus, p65 appears to be a growth-arrest specific gene product whose expression is repressed during the proliferative state of mitotically active mouse kidney cells.Key words: G0 phase, senescence, proliferation, quiescence, SV40-transformed mouse cells.
Developmentally regulated troponin C mRNAs of chicken skeletal muscle
Authors: Berezowsky Craig , Bag Jnanankur
Published: December 2009

Fast and slow/cardiac troponin C (TnC) are the two different isoforms of TnC. Expression of these isoforms is developmentally regulated in vertebrate skeletal muscle. Therefore, in our studies, the pattern of their expression was analyzed by determining the steady-state levels of both TnC mRNAs. It was also examined if mRNAs for both isoforms of TnC were efficiently translated during chicken skeletal muscle development. We have used different methods to determine the steady-state levels of TnC mRNAs. First, probes specific for the fast and slow TnC mRNAs were developed using a 390 base pair (bp) and a 255 bp long fragment, of the full-length chicken fast and slow TnC cDNA clones, respectively. Our analyses using RNA-blot technique showed that fast TnC mRNA was the predominant isoform in embryonic chicken skeletal muscle. Following hatching, a significant amount of slow TnC mRNA began to accumulate in the skeletal (pectoralis) muscle. At 43 weeks posthatching, the slow TnC mRNA was nearly as abundant as the fast isoform. Furthermore, a majority of both slow and fast TnC mRNAs was found to be translationally active. A second method allowed a more reliable measure of the relative abundance of slow and fast TnC mRNAs in chicken skeletal muscle. We used a common highly conserved 18-nucleotide-long sequence towards the 5′-end of these mRNAs to perform primer extension analysis of both mRNAs in a single reaction. The result of these analyses confirmed the predominance of fast TnC mRNA in the embryonic skeletal muscle, while significant accumulation of slow TnC mRNA was observed in chicken breast (pectoralis) muscle following hatching. In addition to primer extension analysis, polymerase chain reaction was used to amplify the fast and slow TnC mRNAs from cardiac and skeletal muscle. Analysis of the amplified products demonstrated the presence of significant amounts of slow TnC mRNA in the adult skeletal muscle.Key words: troponin C mRNA, skeletal muscle, cardiac muscle, polymerase chain reaction, developmental changes in muscle.
Characterization of two forms of cationic peroxidase from cultured peanut cells
Authors: O'Donnell James P. , Wan Lianglu , Huystee R. B. van
Published: December 2009

Two forms of cationic peroxidase from peanut cells were differentiated by concanavalin A affinity chromatography. They differed in molecular mass as well as concanavalin A binding, leading to the initial suggestion that they represented two isozymes of peroxidase. However, similar values for the specific activity, Soret absorption, calcium content, and peptide molecular mass were observed for each of the forms. Therefore, the binding and nonbinding fractions most likely represent two molecular forms of cationic peanut peroxidase, rather than two distinct cationic isozymes. The difference between these two forms is discussed in terms of glycosylation. Through the amino acid sequence analysis of the formic acid treated peptide, the cationic isozyme has been shown to be identical in amino acid sequence to the cDNA clone PNC1.Key words: peanut, peroxidase, glycan, characterization, sequence.
Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins
Authors: Johnstone R. M.
Published: December 2009

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.Key words: transferrin receptor, nucleoside transporter, reticulocyte maturation, multivesicular bodies, 70-kilodalton protein.
Essential fatty acid deficiency lowers the activity of the acetylated low density lipoprotein receptor of rat peritoneal macrophages
Authors: Gavino Grace R. , Levy Emile , Gavino Victor C.
Published: December 2009

We compared phospholipid fatty acid composition, cholesterol ester accumulation, and receptor-mediated binding, internalization, and degradation of acetylated low-density lipoprotein (acetyl-LDL) in cultured peritoneal macrophages from rats fed an essential fatty acid deficient or control diet. The deficient diet increased the 5,8,11-eicosatrienoic acid and decreased the ω6 fatty acid content of macrophage phospholipid relative to control. The deficient diet did not affect macrophage uptake of [1-14C]oleate; however, it lowered the accumulation of intracellular labelled cholesteroyl oleate to 66% of the control. This effect was attributed to a diminution of the specific binding of acetyl-LDL, and not to acetyl-LDL internalization nor to degradation. The results demonstrate the sensitivity of the acetyl-LDL receptor to changes in its membrane environment, brought about through dietary means.Key words: macrophage, acetyl low-density lipoprotein, acetyl low-density lipoprotein receptor, cholesterol, essential fatty acid deficiency.
N-(4-Chlorophenyl)-N-hydroxy-N′-(3-chlorophenyl)urea, a general reducing agent for 5-, 12-, and 15-lipoxygenases and a substrate for their pseudoperoxidase activities
Authors: Falgueyret Jean-Pierre , Desmarais Sylvie , Roy Patrick J. , Riendeau Denis
Published: December 2009

Lipoxygenases contain a nonheme iron that undergoes oxidation and reduction during the catalytic cycle. The conversion from the Fe3+ enzyme form to the Fe2+ form can be achieved using reducing inhibitors, a reaction that can be reversed with lipid hydroperoxides. The present study describes the properties of N-(4-chlorophenyl)-N-hydroxy-N′-(3-chlorophenyl)urea (CPHU), which functions as a reducing agent for various lipoxygenases and stimulates the degradation of lipid hydroperoxide catalyzed by these enzymes (pseudoperoxidase activity). CPHU was a substrate for the pseudoperoxidase reaction of purified soybean lipoxygenase-1 with apparent Km values for CPHU and 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) of 14 and 15 μM, respectively. CPHU was converted during the pseudoperoxidase reaction to a mixture of products that can be resolved by reverse-phase high pressure liquid chromatography. By comparison with the chemical reaction of CPHU and potassium nitrosodisulfonate, the major enzymatic reaction product was tentatively identified as a one-electron oxidation product of CPHU. At low concentrations (50 μM), dithiothreitol completely protected against the degradation of hydroxyurea without inhibiting the pseudoperoxidase reaction. Under these conditions, the rate of the pseudoperoxidase reaction with CPHU as a substrate can be quantitated by the change in absorbance at 234 nm owing to the consumption of 13-HpODE. In addition to soybean lipoxygenase-1, CPHU was found to be a substrate for the pseudoperoxidase activities of purified recombinant human 5-lipoxygenase and porcine leukocyte 12-lipoxygenase. The results are consistent with CPHU reacting with lipoxygenase by a one-electron oxidation to generate the ferrous enzyme form and the nitroxide radical, which could be reduced back to CPHU by DTT. CPHU represents a useful reagent for the preparation of reduced enzyme forms of all types of lipoxygenases and for the spectrophotometric measurement of the pseudoperoxidase reaction in absence of inhibitor oxidation.Key words: lipoxygenase, hydroxyurea, peroxidase, hydroperoxide.
The effect of epidermal growth factor on liver lipid peroxidation and glutathione levels in lobectomized rats
Authors: Gokcora Nahide , Gundogdu Sadi , Aricioglu Aysel , Erbas Deniz , Durmus Osman , Turkozkan Nurten
Published: December 2009

Epidermal growth factor (EGF) is a growth-promoting polypeptide which is found in highest levels in male mice in the submaxillary gland. It may also be a key factor in regeneration of the liver. We performed experiments with 18 male Wistar rats, divided into three groups. Hepatic left lobectomy (%30) was performed on the first group of rats. This group received an intraperitoneal injection of EGF for 7 days. The second group was the control group into which normal saline was injected for 7 days. The third group was sham-operated. On days 5 and 7 tomographic studies of liver were performed. On day 7 EGF levels, lipid peroxidation, and glutathione in liver were measured in all of the rats. While serum EGF levels did not show any significant change, the levels of lipid peroxide were decreased and glutathione was increased. Tomographic measurements indicated that administration of EGF increased the amount of regeneration.Key words: epidermal growth factor, liver lobectomy, lipid peroxide, glutathione, radioimmunoassay.
Visualization of a mammalian transcription initiation complex
Authors: Welsh E. A. , Zahradka P. , Larson D. E. , Sells B. H. , Harauz G.
Published: December 2009

Various proteins required for the initiation of eukaryotic gene transcription by RNA polymerase II have been identified and characterized, but little is known about their organization into a functional unit. Here, we describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by transmission electron microscopy. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was imaged by standard transmission electron microscopy. Quantitative image analysis demonstrated that the complex is a multilobed structure whose two-dimensional projections are approximately 24 × 34 nm in size. Looping of the DNA seen in these images suggests that the proteins residing at the promoter region associate with proteins several hundred base pairs distant to the RNA start site, with bending of the DNA allowing these interactions to occur.Key words: electron microscopy, ribosomal protein gene, gene transcription, transcription factors.
Inhibitors of human immunodeficiency virus-1 protease
Authors: Tyagi Suresh C.
Published: December 2009

We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) can be characterized by a high-affinity mode (Ki = 478 ± 27 nM), resulting in pure competitive inhibition of the hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 μM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 ± 1 μM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 μM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease.Key words: HIV-1 protease, inhibitors, pepstatin, haloperidol, tryptophan fluorescence.
Growth of HeLa S3 cells cotransfected with plasmids containing a c-fos gene under the control of the SV40 promoter complex, pRSVcat, and G418 resistance
Authors: Pai Suresh R. , Bird Richard Curtis
Published: December 2009

HeLa S3 cells, which have been fractionated into sequential and synchronous cell cycle phase-specific fractions, express c-fos at twice the basal levels in the earliest part of G1 phase. To determine whether this peak in c-fos synthesis has regulatory significance, a DNA construct was prepared which contained the human c-fos gene under the transcriptional control of the SV40 promoter complex. The pc-fos(human)-1 gene (9 kilobases) was inserted into the eukaryotic expression vector pSG5 (4.076 kilobases) at the EcoRI site. Electroporation with an exponentially decaying pulse was employed to cotransfect this construct into HeLa S3 cells along with the plasmids pRSVcat and the neomycin-resistance plasmid pFβfos3′ neo. The level of transient expression of each plasmid was determined. Transfection efficiency was determined as percentage fluorescent cells by measurement of immunofluorescence with a chloramphenicol acetyltransferase (CAT) antibody. Efficiency of transfection ranged up to approximately 5% of the cells. Transfected cells were selected on the basis of resistance to Geneticin (G418) at 400 μg/mL. CAT fluorescence and Geneticin resistance were employed to select permanently transformed cell lines. Compared with exponentially growing cells, successfully transfected cell lines expressed more than twice the level of c-fos mRNA as determined by dot-blot analysis and 16 times more of the 62-kilodalton c-fos protein as determined by Western blot analysis. As all cells in the population were not stable c-fos transfectants, this value is likely to be an underestimate of the overexpression level. In addition, expression was under the control of a strong serum induction insensitive promoter, unlike the native c-fos promoter. These transfected cell lines should be useful in determining what regulatory role c-fos plays in reentry into the cell cycle from quiescence as well as during the exponential cell cycle.Key words: c-fos, HeLa, plasmid, SV40 promoter, pRSVcat, RSfos, G418.
New developments in the synthesis of phosphopolyprenols and their glycosyl esters
Authors: Shibaev V. N. , Danilov L. L.
Published: December 2009

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of α- and β-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.Key words: polyprenyl phosphates, polyprenyl phosphosugars, chemical phosphorylation, specificity of enzymes.
Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings
Authors: Rymerson Robert T. , Carroll Kenneth K. , Rip Jack W.
Published: December 2009

Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein∙h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein∙h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds.Key words: dolichol, polyprenol, dolichyl phosphate, polyprenyl phosphate, kinases.
Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids
Authors: Schutzbach John S. , Zimmerman Janet W.
Published: December 2009

The activity of purified recombinant yeast dolichyl-phosphomannose synthase (EC 2.4.1.83) was assessed following reconstitution of the enzyme with phospholipids. The yeast synthase, similar to the mammalian enzyme, was active when reconstituted with phosphatidylethanolamine dispersions but had little (less than 5%) activity in stable phosphatidylcholine bilayers. The enzyme was activated by adding increasing amounts of diacylglycerol to phospholipid bilayers, suggesting that activity of the yeast enzyme was dependent on lipid phase properties rather than specific phospholipids. The synthase could also be reconstituted as an active enzyme in bilayers prepared with a commercial crude lipid preparation containing 40% phosphatidylcholine, but at a rate 10% of that occurring in phosphatidylethanolamine. Vesicles composed of the 40% phosphatidylcholine lipid mixture, dolichyl phosphate, and enzyme were leaky in the presence of divalent cations, and dolichyl-phosphomannose synthase did not appear to catalyze the translocation of dolichyl phosphomannose across membranes at a catalytically significant rate under the assay conditions employed.Key words: dolichyl phosphate, mannose, synthase, transport, liposomes.
Protein-linked isoprenoid lipids in dexamethasone-treated human lymphoid lines in culture
Authors: Bansal Neelam , Nyquist David A. , Melnykovych George
Published: December 2009

Accumulation of isoprenoids was studied in two cell lines derived from acute T-cell leukemia: CEM-C7 cells, whose growth is inhibited by the glucocorticoid dexamethasone, and CEM-C1 cells, which are resistant to this steroid. Isoprenoids were measured by growing the cells in serum-free medium in the presence of lovastatin, which blocks synthesis of mevalonate, and then labeling with exogenous [3H]mevalonolactone. In both cell lines, isoprenoids associated with proteins were detected in cytoplasm, nucleus, and chromatin, and in the chromatin residue that remains after extraction of histone and nonhistone proteins. Differences in labeling were detected after treatment with dexamethasone in the CEM-C7 line, showing a decrease in the cytoplasmic fraction with a corresponding increase in both the nuclear and chromatin fractions as compared with untreated cells. No change was seen in the CEM-C1 line. In both cell lines, 25–30% of the incorporated label was released by treatment with acid or alkali. However, the majority of the label required treatment with methyl iodide for the release of organic-soluble tritiated products. After extraction with chloroform, the lipid fractions contained farnesol, geraniol, dolichols, and possibly nerolidol.Key words: isoprenoids, dexamethasone, leukemia, cell culture, prenylation.
Dolichol and N-linked oligosaccharide synthesis in the rat testis: interaction between Sertoli and spermatogenic cells, evidence for paracrine effects
Authors: Page Kathleen Creed , Mason Paul B. , Lindstrom Lynn , Swan James S. , Nyquist Sally E.
Published: December 2009

The relative contribution of the Sertoli cell and the pachytene spermatocyte to dolichol and N-linked oligosaccharide biosynthesis within the seminiferous tubule was investigated. Evidence is presented to show that the interaction between these two cell types affects dolichol and N-linked oligosaccharide biosynthesis. Analysis of the dolichol content of Sertoli cultures confirms earlier data suggesting that the Sertoli cell constitutes the major pool of dolichols within the seminiferous tubule. [14C]Acetate incorporation studies suggest that the Sertoli cell in culture synthesizes dolichol much more rapidly than does the isolated pachytene spermatocyte. This information, in addition to previous data in the literature, infers an interactive effect whereby the presence of the spermatogenic cell in the tubule stimulates dolichol synthesis in the Sertoli cell. The absence of normal Sertoli-spermatocyte interactions in in vitro incubations may also limit dolichol synthesis in the pachytene spermatocyte. The distribution of dolichol kinase between the Sertoli and the pachytene spermatocyte was also examined. The concentration of this enzyme in the Sertoli cell suggests the presence of an active salvage pathway within that cell. The correlation between the appearance of the pachytene spermatocyte and the previously described peak of dolichol kinase activity in the seminiferous tubules of the prepubertal animal implies cell–cell interactions. Radiolabelling studies of N-linked oligosaccharides were conducted using [3H]mannose and concanavalin A affinity chromatography to identify multiantennary, biantennary, and high-mannose oligosaccharide pools. An in vitro bicameral coculture system was used to demonstrate that pachytene spermatocytes stimulate incorporation of [3H]mannose into Sertoli cell oligosaccharides. The presence of spermatocytes also induced a shift of label from the multiantennary oligosaccharide pool to the high-mannose pool in the Sertoli cell. Reciprocal experiments, in which the pachytene spermatocyte oligosaccharide pools were observed, showed no significant changes. These studies show a clear pachytene spermatocyte derived paracrine effect on Sertoli cell glycosylation.Key words: glycoprotein, dolichol, Sertoli, spermatocyte.
Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase
Authors: Vardanis Alexander
Published: December 2009

The glycogen particle – glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.Key words: glycogen particle, glycogenolysis, glycogenesis, glycogen phosphorylase, glycogen synthase.