bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells
Authors: Zha Xiliang , Jay Francis T. , Choy Patrick C.
Published: December 2009

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labellings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labellings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labelling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.Key words: choline uptake, phosphatidylcholine biosynthesis, amino acids, ethanolamine, BHK-21 cells.
Fatty acid uptake in Escherichia coli: regulation by recruitment of fatty acyl-CoA synthetase to the plasma membrane
Authors: Mangroo Dev , Gerber Gerhard E.
Published: December 2009

Fatty acid uptake in Escherichia coli has been shown to be inhibited by starvation and to be reversed by a short preincubation of the starved cells with D- or L-lactate, succinate, and acetate; these effects on oleate uptake were due to regulation of the rate-limiting step which involves fatty acyl-CoA synthetase. Investigation into the mechanism of regulation of fatty acyl-CoA synthetase showed that D-lactate did not affect the activity of the enzyme directly. Fatty acyl-CoA synthetase was found to be activated by about 20-fold by Triton X-100 and by another 4-fold by the addition of bacterial membranes. D-Lactate treatment was shown to result in coisolation of fatty acyl-CoA synthetase with the plasma membrane; these results are consistent with the interpretation that recruitment of the enzyme to the plasma membrane by D-lactate results in its activation and consequently in the increased level of fatty acid uptake.Key words: fatty acid, uptake, regulation, recruitment, fatty acyl-CoA synthetase, Escherichia coli, plasma membrane.
Modulation of actin polymerization by an exogenous protein, lysozyme
Authors: Kuckel Charles Lee , Lambooy Peter K. , Farnsworth Patricia N.
Published: December 2009

Several methods (fluorescence, high- and low-shear viscosity, and electron microscopy) have been applied to measure the effects of lysozyme on actin polymerization. Under our conditions, at pH 8.0 and 20 °C, lysozyme is predominantly dimeric and its major effect is to inhibit the steady-state polymerization of actin. Those actin filaments formed in the presence of lysozyme are significantly shortened with recurrent amorphous densities along the filament length. However, at pH 6.4 and 37 °C, lysozyme is monomeric and actin filament cross-linking is observed. We reasoned that in hen egg white lysozyme the tripeptide L-arginyl-glycyl-aspartate (RGD), a sequence capable of mimicking a portion of the receptor sites of extracellular matrix proteins, might be important in lysozyme self-association and, therefore, actin-lysozyme interaction. The presence of RGD in the lysozyme-actin polymerizing solutions at pH 8.0 and 20 °C caused an inhibition of the dimeric lysozyme effects, while RGD alone had no effects on actin polymerization. Therefore, RGD most likely binds to a complementary RGD sequence on lysozyme and alters its ability to interact with actin and modify polymerization.Key words: lysozyme, polymerization, dimerization, fluorescence.
Activation parameters for the reconstitution of apotyrosinase by copper
Authors: Wigfield Donald C. , Goltz Douglas M.
Published: December 2009

The reaction of apotyrosinase with divalent copper to give enzymatically active tyrosinase has been studied at pH 8.2 and temperatures from 278 to 303 K. At a 10-fold excess of Cu(II) over enzyme, the pseudo-first order rate constants range from 1.32 × 10−3 s−1 to 2.93 × 10−2 s−1 and yield activation parameters of ΔH≠ = 85 ± 3 kJ∙mol−1 and ΔS≠ = 5 ± 20 J∙mol−1∙K−1. The near zero value for the entropy of activation is discussed.Key words: tyrosinase, copper.
Ontogenic changes in hepatic metallothionein isoforms in prenatal and newborn rats
Authors: Chan Hing Man , Cherian M. George
Published: December 2009

The relative quantities of the two metallothionein isoforms (MT-1 and MT-2) in prenatal and newborn rat livers have been quantified using a competitive enzyme-linked immunosorbent assay with two specific polyclonal antisera. Both MT isoforms were detected in fetal liver on gestation day 16 and their levels continued to increase into the neonatal period. A concomitant increase was observed in plasma MT levels in the pregnant rats. The level of MT-2 was higher (75%) than that of MT-1 in the liver of both prenatal and neonatal rats. Both hepatic MT isoforms showed a significant correlation (P < 0.05) with the hepatic Zn level. Immunohistochemical localization of MT isoforms showed no apparent difference in intracellular distribution. Both MT isoforms were mainly cytoplasmic in fetal rat liver. Intense intranuclear staining was observed in newborn rat liver when the total MT concentration reached a maximum of over 1 mg MT/g tissue. The nuclear MT staining decreased with age, and at day 7, approximately equal concentrations of MT were found in nucleus and cytoplasm. When the newborn rats (1 day old) were injected with Zn (10 mg/kg), both MT isoforms were induced on days 2 and 7. Injection of Cd (1 mg/kg) increased the hepatic MT levels only on day 7. The level of MT-2 remained higher than that of MT-1 in all cases. These results suggest that both MT isoforms play a similar role in the metabolism of Zn during early development and the relative abundance of the MT isoforms may be a species-specific phenomenon.Key words: ontogeny, metallothionein, isoforms, immunohistochemistry, metals.
The polypeptide components of scintillons, the bioluminescence organelles of the dinoflagellate Gonyaulax polyedra
Authors: Desjardins Michel , Morse David
Published: December 2009

Scintillons, the bioluminescence organelles of Gonyaulax polyedra, were purified by isopycnic density gradient centrifugation until only low levels of contaminating chloroplasts and mitochondria were detected by fluorescence and electron microscopy. Purified scintillons catalyzed the luminescent reaction with kinetics identical to those observed during the bioluminescence flash in vivo. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the organelles appeared to contain only two proteins. These proteins were identified as luciferase (135 kilodaltons) and luciferin-binding protein (75 kilodaltons) based on their size and their known functions in the bioluminescence reaction in vitro. The staining of luciferin-binding protein by Coomassie blue was 2.4 ± 0.3 (n = 19) times greater than the luciferase, suggesting that there are four binding protein monomers for every luciferase monomer. A model is proposed for the close packing of the two proteins inside the scintillons.Key words: luciferase, luciferin-binding protein, density gradient centrifugation, dinoflagellate.
Regulation of c-jun/AP-1 expression in rat L6 myoblasts
Authors: Thinakaran Gopal , Bag Jnanankur
Published: December 2009

Myogenic differentiation of skeletal myoblasts in culture is triggered by withdrawal of serum mitogens. Expression of one of the serum-inducible, immediate early genes, the protooncogene c-jun, is maintained under low-serum conditions during myogenic differentiation of L6 myoblasts. In this report we have used agents that modulate protein kinases and Ca2+ levels to investigate how the expression of c-jun and myogenin mRNA and also the activator protein 1 (AP-1) DNA-binding activity are regulated in differentiating L6 cells. Our results show that expression of c-jun and myogenin are regulated independent of each other. Furthermore, down regulation of c-jun expression does not cause an increase in myogenin expression, suggesting that c-jun does not suppress myogenin expression in these cells. Electrophoretic mobility shift and ultraviolet cross-linking analyses revealed that the AP-1 complexes of proliferating myoblasts and differentiating myotubes are formed of similar set of polypeptides, and the AP-1 binding activity is probably modulated by posttranslational modifications in differentiating L6 cells.Key words: myogenin, protein kinase C, cAMP, Ca2+, muscle differentiation.
Microinjection into crane-fly spermatocytes
Authors: Czaban B. Barbara , Forer Arthur , Wise Dwayne A.
Published: December 2009

We were successful in microinjecting fluorescently labelled material into crane-fly spermatocytes. In our experiments, we obtained four results. (i) In most attempts, the membrane stretched around the micropipette and prevented entry of fluorescent material, even when the micropipette appeared to be pushed completely through the cell. This confirms suppositions from earlier micromanipulation experiments that the elastic membrane prevents the micropipette needle from entering the cell, (ii) In some attempts, cells lysed upon contact with the micropipette, (iii) In other attempts, we successfully injected fluorescent material into cells. (iv) Fluorescent material left the cells after injection, often passing into adjacent cells. Although our success rate is low, microinjection into crane-fly spermatocytes is indeed possible.Key words: microinjection, crane-fly spermatocytes.
Substrate- and effector-induced conformational changes in phosphofructokinase from white muscle of rainbow trout (Oncorhynchus mykiss): a fluorescence study
Authors: Su Jeffrey Y. , Storey Kenneth B.
Published: December 2009

Results of activity and spectral studies using fluorescence show that AMP and fructose 2,6-bisphosphate (F2,6P2) activate muscle phosphofructokinase (PFK) from rainbow trout (Oncorhynchus mykiss) through specific and similar conformational changes. Inorganic compounds, such as ammonium and phosphate ions, also increase enzyme activity allosterically; however, the structural alterations in the enzyme caused by these effectors are quite different from those caused by AMP and F2,6P2. No effects of the inorganic compounds on the environment of tryptophan residues of the enzyme were observed. Mg-ATP, a substrate of the enzyme, acts as an allosteric inhibitor at high concentrations. Although Mg-ATP and citrate inhibit the enzyme activity in a synergistic way, the conformational effects of these negative effectors are different. Mg-ATP caused a drastic decrease in fluorescence intensity of the enzyme, whereas citrate did not.Key words: 6-phosphofructo-1-kinase, tryptophan fluorescence, fructose 2,6-bisphosphate, fish muscle glycolysis.
Expression of the protooncogene c-jun is maintained during myogenic differentiation in rat L6 myoblasts
Authors: Thinakaran Gopal , Bag Jnanankur
Published: December 2009

Skeletal myoblasts undergo terminal differentiation when maintained under low-mitogen conditions. We have examined the expression of c-jun, one of the growth-factor-inducible immediate-early genes, during myogenic differentiation of L6 myoblasts. The steady-state levels of c-jun mRNA, c-Jun polypeptide, and activator protein 1 binding activity were not markedly altered in L6 cells undergoing myogenic differentiation. Although expression of c-jun is induced by serum mitogens in fibroblasts and other cell lines, addition of high serum to proliferating myoblasts resulted in the activation of another immediate early gene junB, but not c-jun mRNA expression. These results indicate that regulation of c-jun may differ from that of other immediate early genes in L6 cells. Manipulation of myogenesis by exposing L6 cells to dimethyl sulfoxide also suggested that expression of myogenin and muscle differentiation could occur in the presence of high levels of c-Jun. Furthermore, expression of c-jun from Moloney murine leukaemia viral long-terminal repeat in transfected L6 cells confirmed that constitutive expression of c-jun does not interfere with myogenesis in L6 myoblasts. Therefore, regulation of c-jun expression in rat L6 cells differs from that in the mouse C2 cell line.Key words: myogenesis, c-jun, protooncogene, differentiation, transcription factor.
Initial characterization of endothelial mitogens produced by bovine corpora lutea from the estrous cycle
Authors: Grazul-Bilska Anna T. , Redmer Dale A. , Killilea S. Derek , Zheng Jing , Reynolds Lawrence P.
Published: December 2009

To further characterize mitogenic factor(s) present in luteal extracts or luteal explant conditioned media (LCM), bovine corpora lutea (CL) were homogenized or incubated in explant culture, respectively. After evaluation of luteal extracts and LCM by using an endothelial cell proliferation bioassay, mitogenic activity was characterized by immunoneutralization with antibodies against heparin-binding (fibroblast) growth factor (HBGF) 1 or 2. LCM also were subjected to ultrafiltration, as well as anion-exchange, cation-exchange, and heparin-affinity chromatography. The presence of HBGF-2 in LCM also was evaluated by using a dot immunoblot assay. Extracts of luteal tissues and LCM stimulated (P < 0.05) proliferation of endothelial cells in a dose-dependent manner. Mitogenic activity of luteal extracts and LCM was decreased (P < 0.05) by treatment with specific antibodies against HBGF-2 or HBGF-1. LCM also contained immunoreactive HBGF-2. The mitogenic activity bound to anion exchangers, phenyl-Sepharose, and heparin–agarose, but not to cation exchangers, indicating that endothelial mitogenic activity is anionic at neutral pH, has some hydrophobic characteristics, and belongs to the HBGF family of proteins. Following ultrafiltration, endothelial mitogenic activity was retained by membranes having a 30 000 or 100 000 molecular weight cutoff. In addition, LCM was resolved into four peaks of heparin-binding endothelial mitogenic activity, each with a different affinity for heparin. These data demonstrate that bovine CL contain and produce endothelial mitogens of large molecular size, which may be important regulators of luteal function. These endothelial mitogens are heparin-binding and anionic at neutral pH. In addition, a large portion of the endothelial mitogenic activity produced by bovine CL appears to be immunologically related to HBGF-2 (basic fibroblast growth factor).Key words: angiogenesis, endothelial mitogens, corpus luteum, bovine.
Identification of high density lipoprotein binding proteins in mature adipocyte plasma membranes
Authors: Shen Xin-Yi , Angel Aubie
Published: December 2009

High density lipoprotein (HDL) binding proteins were identified in nonreduced detergent extracts of plasma membranes or crude membrane fractions of rat adipocytes by ligand blotting. Using 125I-labelled human apolipoprotein-E-free HDL ([125I]HDL3), two binding proteins in adipocyte membranes were detected with apparent molecular masses of 122 and 88 kilodaltons (kDa), respectively. The binding of HDL3 to both binding proteins was abolished by pronase treatment and was inhibited by excess unlabelled HDL3. Excessive unlabelled low density lipoprotein reduced the binding of [125I]HDL3 to the 122-kDa binding protein relatively less than that to the 88-kDa binding protein. Polyclonal antisera against purified rat apolipoprotein A-I (apoA-I) effectively inhibited the binding of HDL3 to adipocyte membranes. Affinity-purified antibodies against rat apoA-I also revealed two HDL-binding proteins in rat adipocyte and liver plasma membranes preincubated with rat HDL. The sizes of the HDL-binding proteins in adipocyte plasma membranes detected by anti-apoA-I were similar to those detected by radiolabelled ligand blotting and their counterparts in rat liver plasma membranes. The study demonstrates two HDL-binding proteins, distinguishable by apparent molecular masses and ligand binding affinity, in plasma membrane proteins of mature rat adipocytes using radiolabelled ligand and immunoligand blotting techniques. The results suggest that apoA-I is involved in the interactions between HDL and both variants of HDL-binding proteins.Key words: high density lipoprotein binding proteins, rat adipocytes, apolipoprotein A-I.
Relationship between adenosine 3′,5′-cyclic monophosphate level, cell proliferation, and α-amylase in rat parotid
Authors: Prasad Kedar N. , Carvalho Erika , Kumar Sanjay , Edwards-Prasad Judith , Rosa Francisco G. La
Published: December 2009

Treatment of rats with isoproterenol (IPR, β1- and β2-agonist), dobutamine (β1-agonist), and terbutaline (β2-agonist) increased the cAMP level and cell proliferation, but it decreased α-amylase activity in parotid glands. Terbutaline was relatively less effective.α-Amylase activity as determined by substrate hydrolysis was maximally depressed (85% of control) after 15 min of IPR treatment; however, α-amylase level as determined by Western immunoblotting with the primary antibody to α-amylase decreased maximally after 2 h of treatment. The level of α-amylase significantly decreased after 15 min of treatment, but the levels of α-amylase mRNAs of 702 and 1176 base pairs (bp) slightly increased at this time. This suggests that an increased rate of transcription or a decreased rate of degradation of α-amylase mRNAs without a proportional increase in the rate of translation of α-amylase occurs in parotid glands after treatment with IPR. The levels of α-amylase mRNAs decreased after 2 h of treatment; however, the level of α-mRNA of 702 bp continued to decline, but the level of α-mRNA of 1176 bp increased at 3 days after IPR treatment. The level of actin mRNA of 2200 bp increased at 15 min and remained elevated during the entire observation period. Results showed that IPR-induced elevation of cAMP increases cell proliferation, and alters the level and activity of α-amylase and the expression of α-amylase genes at different rates.Key words: parotid, α-amylase gene, cell proliferation, β-receptor.
Purification and kinetic characterization of pickerel liver alcohol dehydrogenase with dual coenzyme specificity
Authors: Al-Kassim Loola S. , Tsai C. Stan
Published: December 2009

A major alcohol dehydrogenase isozyme that displays dual coenzyme specificity has been purified from pickerel liver by ion-exchange, gel filtration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dimeric and possesses common physical properties shared by other liver alcohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectroscopy demonstrates that NADP+ binds to two coenzyme sites of the pickerel enzyme. Steady-state kinetic studies suggest that pickerel liver alcohol dehydrogenase catalyzes NAD(P)+-linked ethanol oxidation via a random pathway. While the NADP+ reduction involves the formation of an abortive complex at high NADP+ concentrations, the NAD+ reduction at low NAD+ concentrations follows an ordered Bi-Bi mechanism with NAD+ being the leading reactant.Key words: purification, pickerel liver, alcohol dehydrogenase.
Multifunctional activities of pickerel liver alcohol dehydrogenase
Authors: Tsai C. Stan , Al-Kassim Loola S.
Published: December 2009

A major isozyme of pickerel liver alcohol dehydrogenase has been purified to homogeneity. The enzyme, in addition to catalyze NAD(P)+-linked dehydrogenation of alcohols, also mediates dismutation of aldehydes and hydrolysis of esters. Steady-state kinetic studies and chemical modifications of the pickerel liver enzyme with respect to its esterolytic and dismutative activities were carried out. Pickerel liver alcohol dehydrogenase catalyzes hydrolyses of p-nitrophenyl esters via a Uni-Bi mechanism, with alkanoic acids as the last product released. Modifications of Cys and Lys suppress the esterolytic activity. A random mechansim with the formation of dead-end complexes is implicated for the dismutation of octanal catalyzed by pickerel liver alcohol dehydrogenase. Two amino acid residues, Cys and His, are involved in the dehydrogenation as well as dismutation reactions. The present study identifies a regulatory function of Lys for the multifunctional activities of liver alcohol dehydrogenase. When the Lys residue is specifically glucosylated, the dehydrogenase activity increases. Its esterase activity decreases, while the dismutase activity remains unchanged.Key words: multifunctionality, pickerel liver, alcohol dehydrogenase.
Characterization of the gene encoding the glutamic-acid-specific protease of Streptomyces griseus
Authors: Sidhu Sachdev S. , Kalmar Gabriel B. , Borgford Thor J.
Published: December 2009

The complete gene sequence (sprE) for the glutamic-acid-specific serine protease (SGPE) of the gram-positive bacterium Streptomyces griseus is reported. The sprE gene encodes a 355 amino acid pre–pro–mature enzyme. The presence of a glutamic acid residue at the junction of the pro and mature segments of the protein suggests that the enzyme is self-processing. SGPE was found to have extensive homology with the S. griseus proteases A and B. However, there is an additional segment to the pro region of SGPE, lacking in proteases A and B, which has significant homology to the pro region of the α-lytic protease of the gram-negative bacterium Lysobacter enzymogenes. Expression of recombinant SGPE in Bacillus subtilis is also reported, and the enzyme is shown to be self-processing.Key words: serine protease, maturation, expression, propeptide.
Regulation of low density lipoprotein receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities are differentially affected in Niemann–Pick type C and type D fibroblasts
Authors: Sidhu Harkirat S. , Rastogi Stella A. R. , Byers David M. , Guernsey Duane L. , Cook Harold W. , Palmer Frederick B. St. C. , Spence Matthew W.
Published: December 2009

Defective regulation of intracellular cholesterol metabolism has been investigated in cultured fibroblasts from two subtypes of Niemann–Pick type II disease: the panethnic Niemann–Pick type C (NPC) and the Nova Scotia type D (NPD). Cell extracts from NPC and NPD fibroblasts cultured in lipoprotein-deficient medium exhibited activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that were two-fold greater than in normal cells. Addition of serum resulted in only a 15% decrease in HMG-CoA reductase activity within 6 h in these cells, compared with a decrease of 80% in normal fibroblasts. The initial rate of return to maximal values for the first 6 h after removal of serum was similar in all three cell types; thereafter, the rate was faster in the mutant fibroblasts. Binding and internalization of 125I-labeled low density lipoprotein (LDL) was not decreased within 12 h of incubation of NPC fibroblasts with serum, while a decrease of 50% was observed for both NPD and normal fibroblasts over this time period. Northern blot analysis also indicated a slower decrease in steady-state LDL receptor mRNA in NPC relative to normal and NPD cells. In all three cell types, inhibition of HMG-CoA reductase with mevinolin had no effect on serum-stimulated cholesterol esterification, while inhibition of acyl-CoA:cholesterol acyltransferase with Sandoz 58-035 did not influence HMG-CoA reductase activity, indicating that defects in these regulatory mechanisms are independent. Together with previous observations that NPC and NPD fibroblasts exhibit different levels of cholesterol accumulation and esterification, our results suggest that NPD may result from a distinct mutation in a Niemann–Pick type II gene, in which different mutations can differentially alter the various mechanisms of cholesterol regulation.Key words: Niemann–Pick disease, cholesterol regulation, low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase.
Aldehyde dehydrogenase and cytotoxicity of purified bovine serum amine oxidase and spermine in Chinese hamster ovary cells
Authors: Averill-Bates Diana A. , Agostinelli Enzo , Przybytkowski Ewa , Mondovi Bruno
Published: December 2009

Bovine serum amine oxidase (EC 1.4.3.6) catalyses the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia, and hydrogen peroxide. It has been suggested that the dialdehyde produced during the oxidation of spermine subsequently undergoes spontaneous β-elimination to form acrolein. Oxidation of the aldehydes by aldehyde dehydrogenase (EC 1.2.1.5) thus eliminates these reactive species and prevents the formation of acrolein. This work studies the role of each of the oxidation products of spermine in cytotoxicity induced by purified bovine serum amine oxidase. The inhibition patterns of NAD-dependent aldehyde dehydrogenase and catalase against cytotoxicity of bovine serum amine oxidase were determined in Chinese hamster ovary cells at 37 °C. Cytotoxicity caused by exogenous hydrogen peroxide, added directly (> 10 μM) or generated by glucose oxidase (0.5 U/mL), was completely inhibited by catalase. Cytotoxicity caused by bovine serum amine oxidase (5.7 × 10−3 U/mL) and spermine (340 μM) was completely inhibited by catalase only during short incubation times after which time cytotoxicity occurred. This indicates that hydrogen peroxide was the only species contributing to cytotoxicity at this stage of the reaction. Aldehyde dehydrogenase alone caused partial inhibition of cytotoxicity, but only later in the reaction. Cytotoxicity was completely eliminated in the presence of both catalase and aldehyde dehydrogenase. Exogenous acrolein (> 50 μM) also caused cytotoxicity in Chinese hamster ovary cells. However, hydrogen peroxide was toxic to cells at lower concentrations and at shorter exposure times relative to aldehydes. These data show that both peroxide and aldehydes contribute to cytotoxicity of oxidation products of spermine. Aldehydes such as acrolein are responsible for cytotoxicity that cannot be accounted for by hydrogen peroxide.Key words: acrolein, hydrogen peroxide, catalase, polyamine.
Nuclear magnetic resonance studies of the structure of B50/neuromodulin and its interaction with calmodulin
Authors: Zhang Mingjie , Vogel Hans J. , Zwiers Henk
Published: December 2009

B50/neuromodulin is a neuronal phosphoprotein that is found in association with the inner membrane of nerve cells. In this work, we have studied the structure of bovine B50 in aqueous solution (pH 7.5) by 1H nuclear magnetic resonance (NMR) spectroscopy and our results indicate that B50 is an unstructured protein under these conditions. One-dimensional 1H-NMR titration studies of the interaction between B50 and calmodulin (CaM) have shown that B50 does not interact with (or) interacts very weakly with apo-CaM in solution; neither does B50 interact with Ca2+-CaM. These NMR data are consistent with an earlier observation that B50 is not capable of binding apo-CaM in vitro unless some nonionic detergent is present. We have also detected aromatic NMR peaks for a new posttranslational modification that might involve the His residues of the protein. The interaction of a 14-residue peptide (I38-L51) encompassing the CaM-binding domain of B50 with CaM was also studied by NMR. We have found from two-dimensional transferred nuclear Overhauser enhancement experiments that the B50 peptide binds weakly to apo-CaM in an α-helical conformation; the α-helix appears to be induced by the binding of the peptide to apo-CaM.Key words: calmodulin, protein B50, neuromodulin, two-dimensional NMR, α-helix.
A low molecular weight peptide from snow mold with epitopic homology to the winter flounder antifreeze protein
Authors: Newsted W. Jay , Polvi Sandra , Papish Bob , Kendall Ed , Saleem Mohammed , Koch Muffy , Hussain Atta , Cutler Adrian J. , Georges Fawzy
Published: December 2009

Evidence for a small size protein (ca. 3500 kDa) exhibiting epitopic homology to the Atlantic winter flounder antifreeze protein (AFP) is found in the snow molds Coprinus psychromorbidus, Myriosclerotinia borealis, and Typhula incarnata. The protein shows strong cross-reactivity with antisera specific for the flounder AFP. Preliminary studies suggest that the protein is synthesized in response to lowering the culture temperature, and that it is membrane associated and, therefore, may function in an analogous capacity to the fish AFP. Also, the protein is shown to have antifreeze properties as determined by nuclear magnetic resonance microimaging experiments.Key words: antifreeze proteins, low temperature stress, snow mold, winter flounder.
Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell
Authors: Arao Makoto , Yamaguchi Toru , Sugimoto Toshitsugu , Fukase Masaaki , Chihara Kazuo
Published: December 2009

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Sue, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from α-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250–300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1–84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.Key words: opossum kidney, parathyroid hormone, chymotrypsin, endopeptidase.
Effect of pH and nonphysiological salt concentrations on human immunodeficiency virus-1 protease dimerization
Authors: Tyagi Suresh C. , Simon Sanford R. , Carter Carol A.
Published: December 2009

Human immunodeficiency virus-1 (HIV-1) protease is catalytically active as a dimer of identical subunits that associate through noncovalent interactions. To investigate the forces stabilizing HIV-1 protease in its active form, we have studied the effects of pH and salts on structure and function of the enzyme. Enzymatic activity was measured by following the hydrolysis of a fluorogenic substrate. Dissociation of the dimer into its subunits was monitored by gel filtration, while conformational changes in the enzyme were probed by measurements of intrinsic tryptophan fluorescence. Mg2+ ions were capable of dissociating the dimeric enzyme with a concomitant red shift and increase in quantum yield of the tryptophan fluorescence, indicating increased accessibility of tryptophan to the aqueous environment. These structural changes also were associated with a loss of catalytic activity which was insensitive to substrate concentration, consistent with noncompetitive inhibition. Both structural and functional changes could be attributed to binding of Mg2+ ions to a site with an apparent dissociation constant of ~2 M. In contrast, increasing concentrations of Na ions up to 5 M were without effect. Increasing pH had similar effects on HIV-1 protease as increasing Mg2+ ions concentration, with concomitant dissociation into subunits, increase in quantum yield and red shift in tryptophan fluorescence, and loss in catalytic activity. The apparent pKa for these structural and functional transitions was 6.95 ± 0.08. This value is consistent with that of an aspartic acid residue with an anomalously high pKa, which has been implicated in the catalytic activity of HIV-1 protease.Key words: human immunodeficiency virus-1 protease, dimerization, pH and salt effects.
DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes
Authors: Hamelin Claude , D'Amours Benoit , Page Christian , Chung Young Sup
Published: December 2009

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.Key words: deoxyribonucleoproteins, DNA topoisomerase, minichromosomes, ribonucleoproteins, simian virus 40, viral chromatin.
Kinetics of uptake, transport, and accumulation of liposome-associated fluorescent dolichol esters in human fibroblasts
Authors: Jiang Lianwei , Rip Jack W.
Published: December 2009

The anthroyl and n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) hexanoyl esters of dolichol were synthesized and incorporated into phospholipid liposomes. Fluorescence spectrometric methods were used to estimate the kinetics and dynamics of the incorporation and turnover of these esters in normal human fibroblasts. For anthroyl dolichol a saturable uptake of 2.5 × 103 pmol/106 fibroblasts was obtained. Half-maximum uptake was seen at a labeling concentration of 19 μM. The time required for half-maximum uptake of fluorescence (t1/2) was about 10 h. Over 50% of the anthroyl dolichol taken up remained in fibroblasts 24 h after the labeling medium was removed. Uptake was higher for esters of 9 isoprenes than for those with 16–21. Dolichol labeled with the NBD fluorophore appeared to enter fibroblasts in higher concentration than the same dolichol labeled with anthracene. Uptake was not influenced by the presence of agents that disrupt lysosome function (leupeptin and chloroquine) prior to or during fluorescence labeling. The amount of fluorescent dolichol in the (i) lysosomes and endosomes and (ii) nuclei of labeled fibroblasts was determined after Percoll density gradient centrifugation and cell lysis in culture, respectively. Most of the fluorescent dolichyl ester (and most of the free alcohol form) taken up by fibroblasts was recovered in lysosomes.Key words: anthroyl dolichol, n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoyl dolichol, lysosome, localization, laser scanning confocal microscopy, turnover.
Casein-derived peptides can modulate the production of 5-hydroxyeicosatetraenoic acid in human neutrophils
Authors: Rabgaoui Najat , Guérin Marie-Christine , Torreilles Jean
Published: December 2009

The effects of peptides derived from bovine casein hydrolysates were evaluated on oxygen-free radical release, 5-hydroxyeicosatetraenoic acid (5-HETE), and leukotriene B4 (LTB4) production by human polymorphonuclear leukocytes (PMNs). PMNs were activated by N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, calcium ionophore (A23187), or opsonized zymosan. Tyrosyl-prolyl-phenylalanyl-proline (fragment 60–63 from bovine β-casein) inhibited oxygen-free radical production and 5-HETE production but had no effect on the production of LTB4 and LTB4 isomers. Arginyl-tyrosyl-leucyl-glycyl-tyrosyl-leucine (fragment 90–95 from bovine α-casein) enhanced the production of oxygen-free radical and 5-HETE but had no effect on the production of LTB4 and isomers. Both peptides unsignificantly enhanced the release of arachidonic acid by A23187-stimulated PMNs previously supplemented with [14C]arachidonic acid. Casein-derived peptides then seemed to act on the enzymatic activities of endogenous peroxidases that reduced hydroperoxyeicosatetraenoic acid to 5-HETE. 5-HETE is recognized as an ubiquitous cell function mediator and modulation of PMN 5-HETE production by casein-derived peptides could be implicated in the manifestation of some adverse effects to cow's milk.Key words: human polymorphonuclear leukocytes, casein-derived peptides, oxygen-activated species, 5-hydroxyeicosatetraenoic acid, leukotriene B4.
Substitutions for Gly-794 show that binding interactions are important determinants of the catalytic action of β-galactosidase (Escherichia coli)
Authors: Martinez-Bilbao Mercedes , Huber Reuben E.
Published: December 2009

Substitutions of Gly-794 (β-galactosidase) with Asp, Asn, Glu, and Lys caused decreased binding of substrates and inhibition by substrate analogs, while inhibition by planar and positively charged galactose analogs increased relative to the binding of substrates and the inhibition by substrate analogs. There was a correlation of the relative inhibition with the size of the substituted residue but no relationship to the presence or absence of a negative charge, and as the relative inhibition by the planar and positively charged galactose analogs increased, k3 (hydrolysis; degalactosylation) and kcat/Km (catalytic efficiency) values decreased. The k2 values (glycolytic cleavage; galactosylation) mainly increased for poor substrates (p-nitrophenyl β-galactoside and lactose) but decreased for o-nitrophenyl β-galactoside (a good substrate). Enzymes substituted with Asp or Asn were inhibited to a similar extent by planar and positively charged inhibitors and had similar effects on catalysis, while inhibition and catalytic effects on the enzyme substituted by Glu were quite different. If the negative charge was important, the Asp- and Glu-substituted enzymes should have been inhibited to a similar extent, while the Asn-substituted enzyme should have caused a different degree of inhibition. The enzyme substituted with a Lys at position 794 bound substrates and inhibitors very poorly, but the relative inhibition and the catalysis still correlated to size. Alterations of the size of the residue at position 794 cause modifications in the binding interactions and affected activity. If planar and positively charged galactose derivatives are transition state analogs, they must mimic the transition state for galactosylation (k2) more than the transition state for degalactosylation (k3), since k2 usually increased when the relative inhibition by these inhibitors was better while k3 always decreased. The amounts of Mg2+ required for activation of the substituted enzymes did not correlate with either charge or size.Key words: β-galactosidase, binding, glycine, aspartic, asparagine, glutamic, lysine, mechanism, transition state, catalysis, site-specific substitution.
Catalase activity of chloroperoxidase and its interaction with peroxidase activity
Authors: Sun Weimei , Kadima Tenshuk A. , Pickard Michael A. , Dunford H. Brian
Published: December 2009

The catalatic activity of chloroperoxidase (CPO) was demonstrated to exhibit saturation kinetics under steady-state conditions, which were not observed with catalase under comparable conditions. Results were obtained using reaction mixtures of CPO and H2O2 at pH 6.2, rapid spectral scan and single wavelength measurements, and transient- and steady-state reaction conditions. The observed rectangular hyperbolae (measurement of rates of disappearance of H2O2 and appearance of O2) could be fit quantitatively to[Formula: see text]where v is rate of O2 evolution, [CPO]0 is total enzyme concentration, B1 = (9 ± 1) × 102 s−1, and B2 = (3.3 ± 0.4) × 10−3 M. The results indicated formation of a complex of compound I (CPO-I) and H2O2, which dissociated to native CPO, O2, and H2O with a rate constant of (9 ± 1) × 102 s−1. The determination of the peroxidatic activity of CPO was performed using demethylation of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) under steady-state conditions. Attempts to determine Michaelis–Menten constants for the substrates TMPD and H2O2 gave rise to apparently anomalous data. Our data showed that the modified ping–pong mechanism established for horseradish peroxidase is applicable to the peroxidatic reaction catalyzed by chloroperoxidase. Both peroxidatic and catalatic reactions occurred in the reaction system containing H2O2, a reducing substrate, and CPO. A combined reaction mechanism was proposed for CPO-catalyzed reactions in which the modified ping–pong mechanism was applicable for the peroxidatic reactions and the formation of a CPO-I–H2O2 complex occurred for the catalatic reaction.Key words: chloroperoxidase, catalatic activity, peroxidatic activity, transient-state kinetics, steady-state kinetics.
Characterization of the growth inhibition phenotype of the kilAtelAB operon from IncPα plasmid RK2Ter
Authors: Turner Raymond J. , Weiner Joel H. , Taylor Diane E.
Published: December 2009

The cryptic tellurite resistance (Ter) determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558). The Ter determinant was identified as the kilA locus and comprises an operon of three genes: kilA, telA, and telB (also referred to as klaA, klaB, and klaC on RK2(Tes)). Cultures harboring the plasmid pDT1558 displayed an extended lag phase in rpoS− hosts, but grew normally in rpoS+ strains. Each of the genes from RK2Ter was subcloned into the expression vector pJF118EH behind an inducible tac promoter using polymerase chain reaction (PCR). The PCR primers were used to engineer an efficient ribosome-binding site and an adjacent sequence to improve protein expression. Expression plasmids were modified by inclusion of different resistance markers for selection during complementation. The killing phenotype of the kilAtelAB operon was studied with the overexpressing plasmids. Each individual gene specified growth inhibition of Escherichia coli cells, but with different combinations of the genes giving rise to differing degrees of the inhibition. Additionally, the bacteriostatic and bacteriocidal effects of the genes were found to be different depending on whether or not the cultures were grown in minimal salts medium with glucose or in Luria–Bertani medium. Cells harboring the kilA gene alone or the three genes of the kilAtelAB operon expressed either in trans or cis behind the tac promoter were found to form nonseptated filaments up to 10–30 times the length of control cells. The effect of filamentation was greater for cells grown in minimal salts medium with glucose as the carbon source. This study demonstrates that each of the gene products from kilA, telA, and telB, when expressed at high levels either alone or together with another gene in the operon, exhibit some degree of growth inhibition of host cells. This growth inhibition is considered paramount in the stable maintenance of the plasmid within its host.Key words: RK2, kilA, rpoS, tellurite resistance, subunit complementation, protein overexpression, expression lethality, filamentation.
Molecular biology of rubella virus structural proteins
Authors: Gillam Shirley
Published: December 2009

Rubella virus is a small, enveloped, positive-stranded RNA virus in the Togaviridae family and bears similarities to the prototype alphaviruses in terms of its genome organization and strategy for viral gene expression. Despite being an important human pathogen, the cell biology of rubella virus remains poorly characterized. This review focuses on the molecular biology of rubella virus structural proteins, with emphasis on the proteolytic processing and maturation of virus structural proteins, the glycosylation requirement for intracellular transport and function of glycoproteins, and the localization of hemagglutinin- and virus-neutralizing epitopes. A number of significant differences between rubella virus and alphavirus structural protein expression and maturation were discovered.Key words: rubella virus, N-linked glycosylation, epitope mapping, proteolytic processing.
Apoptosis, retinitis pigmentosa, and degeneration
Authors: Wong Paul
Published: December 2009

The mechanism of photoreceptor cell death in different inherited retinal degenerations is not fully understood. Mutations in a number of different genes (such as rhodopsin, the beta subunit of cGMP phosphodiesterase, and peripherin) have been identified as the primary genetic lesion in different forms of human retinitis pigmentosa, one of the most common causes of inherited blindness. In all cases the manifestation of the disorder regardless of the specific primary genetic lesion is similar, resulting in photoreceptor cell degeneration and blindness. A recent hypothesis is that the active photoreceptor cell death, which is characteristic of these genetically distinct disorders, is mediated by a common induction of apoptosis. In the present review, the current evidence for active cell death during retinal cell death in several different rodent models of retinitis pigmentosa and retinal degeneration is examined.Key words: retinal degeneration, apoptosis, retinitis pigmentosa, clusterin, DNA fragmentation.
Spontaneous apoptosis of cells prepared from the nonregressing corpus luteum
Authors: Kenny Nicholas , Williams Rachel E. , Kelm Lorraine B.
Published: December 2009

At the end of a nonconception estrous cycle, the sheep corpus luteum undergoes involution (luteolysis), a process thought to involve apoptotic deletion of cells. It is not yet clear which of the heterogeneous luteal cell types is involved or what mechanisms drive the apoptotic progression. We examined intact paraffin-embedded corpora lutea (in situ terminal dUTP nick end-labeling method) and found direct evidence for apoptotic deletion of cells during luteolysis, but not in healthy, nonregressing corpora lutea. We then sought to implement in vitro models to dissect apoptotic mechanisms in the constituent cells of the corpus luteum. Cells prepared using standard collagenase dispersion of corpus luteum were evaluated for evidence of apoptosis (DNA laddering) by direct agarose gel electrophoresis, a method that obviates the need for DNA extraction, so allowing examination of relatively few cells (≤ 0.5 × 106). When cells were prepared from nonregressing corpus luteum for in vitro manipulation, a population(s) of cells undergoing spontaneous apoptosis was detected. Apoptosis was inhibited by Zn2+ (5 mM), by the tyrosine phosphatase inhibitor sodium orthovanadate (100 μM), or by maintenance at 4 °C. It appears that simple collagenase digestion of intact corpus luteum removes a subset of constituent cells from their survival signal, leading to rapid initiation of endonuclease activity and apoptotic cell death. Identification of the required survival factors and their actions is being pursued to facilitate development of appropriate in vitro models for this endocrine system.Key words: corpus luteum, apoptosis.