bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyphosphoinositide metabolism in aging human erythrocytes
Authors: Palmer Frederick B. St. C.
Published: December 2009

Normal human erythrocytes were fractionated in a density gradient. Capacity to metabolize polyphosphoinositides was compared in young (least dense) and old (most dense) cells. Polyphosphoinositide synthesis was assessed by following the incorporation of radioactivity from [γ-32P]ATP into the 1-(3-sn-phosphatidyl)-D-myo-inositol 4-phosphate (PtdIns4P) and 1-(3-sn-phosphatidyl)-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P2) of isolated membranes. There was no significant age-dependent change in the ability to synthesize PtdIns4P and PtdIns(4,5)P2 or in the response of the PtdIns and PtdIns4P kinases to Mg2+. The cytosolic Mg2+-dependent PtdIns(4,5)P2 phosphatase was also unaffected by age. The membrane cation-independent PtdIns4P phosphatase activity declined slightly (12%). Therefore, the capacity to catalyse the interconversion among the three phosphoinositides in the membrane is retained throughout the life of the erythrocyte. The Ca2+-dependent polyphosphoinositide phosphodiesterase activity in the membranes was reduced in old cells (57%) to the same extent as the glutamate–oxaloacetate transaminase activity used as an index of cell age. Thus, irreversible loss of polyphosphoinositide from the membrane by the action of this diesterase (prevented in healthy cells by the active maintenance of a very low intracellular Ca2+ concentration) is not very likely even in senescent cells when Ca2+ homeostasis begins to fail.
Affinity chromatography purification and partial characterization of phytohemagglutinin-receptor glycoproteins from porcine splenic lymphocytes
Authors: Dupuis Gilles , Doucet Jean-Pierre , Bastin Bânû , Cardin Jeannine
Published: December 2009

We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA – Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9–2.4% of the amount of protein applied to the column. They contained 20 ± 1.3% hexose and 1.7 ± 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50–55, 75, 95, 130, and 155 kdaltons along with minor bands of 20–40, 42, 45, 60–65, 175, and 200–250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 106 cells/mL in the presence of PHA. A 50% inhibition was observed when 20 μg/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 μg/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line. It is suggested that the affinity-purified PHA-reactive glycoproteins are inhibitors of PHA-dependent cellular responses because they compete with PHA-receptor sites on the lymphocyte plasma membrane. A mouse antiserum raised against the purified glycoproteins inhibited PHA-induced lymphocyte activation, but did not stimulate lymphocytes when added alone to lymphocyte cultures or in combination with a antimouse antiserum.
Measurement of vitellogenin from rainbow trout by rocket immunoelectrophoresis: application to the kinetic analysis of estrogen stimulation in the male
Authors: Maitre Jean Louis , Guellec Catherine Le , Derrien Stephane , Tenniswood Martin , Valotaire Yves
Published: December 2009

The study of the seasonal regulation of vitellogenesis in rainbow trout (Salmo gairdneri) is hampered by two features of the system which are not seen in species such as Xenopus. First, it is impossible to sex immature trout by external appearance and, secondly, the quantitation of the very low levels of vitellogenin in previtellogenic serum is technically difficult and tedious. We describe the preparation of a specific, sensitive anti-vitellogenin antibody and the use of this antibody in a rocket immunoelectrophoresis system to measure serum vitellogenin. The sensitivity of the assay is such that, using only 2 μL of serum, it is possible to detect vitellogenin at levels of 10 μg/mL, making this assay extremely useful for selecting immature female trout for further studies on the basis of the presence of vitellogenin. Using this system we have also measured the response of individual male trout to stimulation with different levels of estradiol, and we have shown that it is possible to measure the stimulation of vitellogenin by estradiol levels equivalent to those seen during the previtellogenic phase of the reproductive cycle in females. This simple assay system thus alleviates two of the major hurdles in studying vitellogenesis in trout.
Size analysis of uncoupling protein and its precursor from brown adipose tissue of different species
Authors: Freeman Karl B. , Meyrick Karen , Patel Hasmukh V. , Ridley Robert G.
Published: December 2009

The apparent size of the monomeric form of uncoupling protein from brown adipose tissue of several mammalian species was compared by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Including earlier results the apparent molecular mass of the protein from rat was about 32 000 daltons and varied about 1500–2000 daltons from the different sources with the size increasing in the order human < rat ≤ mouse ≤ hamster ≤ rabbit. The size of newly synthesized uncoupling protein was also found to vary among species. However in the two cases examined, rat and rabbit, the precursor and its respective mature monomeric protein had the same apparent size as shown by coelectrophoresis.
ATP turnover by the fatty acid reductase complex of Photobacterium phosphoreum
Authors: Rodriguez Angel , Nabi Ivan R. , Meighen Edward
Published: December 2009

A sensitive reverse-phase high pressure liquid chromatographic assay for formation of AMP coupled with analysis of aldehyde production has been used to characterize the properties of the fatty acid reductase complex of Photobacterium phosphoreum. The enzyme complex, which consists of three different polypeptides (34 000, 50 000, and 58 000), has a high affinity for ATP (Km = 20 nM) and shows highest specificity with C14 fatty acids. Activation of the fatty acid is efficiently coupled to the reduction step showing a stoichiometry of one molecule of fatty acid reduced to aldehyde and one molecule of NADPH oxidized for every molecule of ATP converted to AMP. Reconstituted fatty acid reductase (50 000 and 58 000) shows an ATP hydrolase activity that is independent of NADPH with the maximum amount of AMP formed limited by the amount of fatty acid in the assay, consistent with acyl-protein turnover experiments and the channeling of fatty acids to form acyl thioesters (−NADPH) or aldehyde (+NADPH). Addition of the 34 000 polypeptide to the reconstituted enzyme results in stimulation of AMP formation (−NADPH) to a level far exceeding the amount of fatty acid, showing that the fatty acid can be recycled by the 34 000 protein through its thioesterase activity. Also the 34 000 protein is responsible for a two- to three-fold stimulation in the rate of ATP hydrolysis, suggesting that it can be involved in the stabilization of the enzyme complex.
Protein carboxyl methylation–demethylation in rat thymocytes
Authors: Fetters Hazel A. , Kelleher James , Duerre John A.
Published: December 2009

The in vitro carboxyl methylation–demethylation of exogenous and endogenous proteins was investigated in rapidly proliferating thymocytes. Of all the cell fractions tested protein carboxyl methyltransferase activity was highest in the nucleoplasmic fraction (3.2 pmol/mg per minute with ACTH and 0.8 pmol/mg per minute with endogenous substrates). The only other fraction with significant activity was the cytosol (1.2 pmol/mg per minute with ACTH). The nuclei from thymocytes are extremely large; consequently some 70–80% of protein carboxyl methyltransferase in these cells is of nuclear origin. The cellular concentration of protein carboxyl methyltransferase and methyl acceptor proteins paralleled the development of the thymus. Mature lymphocytes contained about 25% of the activity of immature thymocytes. Failure to accumulate protein methyl esters, either in vitro in in vivo, was most likely due to the presence of a very active protein methylesterase. This rapid turnover of protein methyl esters was manifest by the continuous production of [3H]methanol when soluble fractions were incubated with S-adenosyl-L-[methyl-3H] methionine ([methyl-3H]AdoMet). [3H]Methanol production was enhanced upon the further addition of the particulate fractions, particularly chromatin. The turnover of protein methyl esters was primarily enzymatic, since no [3H]methanol was formed when ACTH was incubated with AdoMet and purified protein carboxyl methyltransferase. Utilizing [3H]methanol formation as an index of the rate of protein methylation–demethylation would yield minimal values, since this compound was oxidized via alcohol dehydrogenase or oxidase in these cells. The majority of the methyl acceptor proteins were located in the nuclei. The rapid methylation–demethylation of these proteins may play some role in development and (or) differentiation.
A 19F nuclear magnetic resonance study of the interaction of carbon dioxide with fluoro-amino acids
Authors: Dettman Heather D. , Weiner Joel H. , Sykes Brian D.
Published: December 2009

The 19F nuclear magnetic resonance (NMR) spectra of 3-fiuorophenylalanine and 3-fluorotyrosine in bicarbonate buffer are characterized by a main resonance band and a well-resolved upfield resonance. We show that the upfield resonance is due to the formation of a carbamate between the amino group of the amino acid and bicarbonate. The intensity of the upfield resonance is dependent on the pH (pD) of the solution and reflects changes in the concentration of the free amino acid and carbamate. Thus the normalized spectral peaks can be used to quantitate the free amino acid and carbamate concentrations and these concentrations have been used to determine the equilibrium constant of the carbamate reaction. The value of the equilibrium constant obtained, using an equation which considered the various ionic states of the reactants, was 8.52 × 10−6 ± 0.27 × 10−6 for 3-fiuorophenylalanine and 8.84 × 10−6 ± 0.72 × 10−6 for 3-fluorotyrosine. These values are within the range expected for amino acids and indicate that the fluorine nucleus does not perturb the reaction significantly and that the 19F NMR can be used to quantitate the formation of carbamates in solutions of fluorine-labelled amino acids.
Actin gene number in the sea star Pisaster ochraceus
Authors: Kovesdi Imre , Smith Michael J.
Published: December 2009

The number of actin genes per haploid genome of three sea star species has been measured by reassociation of a single-strand actin-coding sequence probe with excess genomic DNA. Least-squares fit analyses of the reassociation curve indicate five actin genes for Pisaster ochraceus and Pisaster brevispinus and eight actin genes for Dermasterias imbricata. Using 3′ untranslated sequence probes from cDNAs or 3′ noncoding regions of a genomic clone, essentially all of the actin-containing Southern digest fragments can be assigned to the three actin gene classes described earlier for P. ochraceus. Through analyses of genomic digests and dot blots from P. ochraceus probed with both the actin sequence and 3′ sequence, the copy number of gene class members per genome has also been estimated.
Structural investigation of the lipopolysaccharide core isolated from a virulent strain of Vibrio ordalii
Authors: Banoub Joseph H. , Hodder Howard J.
Published: December 2009

The structure of the core oligosaccharide of Vibrio ordalii has been investigated. The studies involved the use of nuclear magnetic resonance, methylation analysis, partial hydrolysis with hydrochloric acid, nitrous acid deamination, partial hydrolysis with sulfuric acid, Smith degradation, and oxidation with chromium trioxide. As a result of these studies the following structure is proposed.[Formula: see text]
Isolation and characterization of nuclear envelopes from three variant cell lines of the Shionogi mouse mammary carcinoma: Identification of androgen-dependent peptides
Authors: Golsteyn Elizabeth J. , Po Cecilia , Lefebvre Yvonne A.
Published: December 2009

We have isolated and purified, with good yields, nuclear envelopes from an androgen-responsive and from two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma after subjecting purified nuclei to DNase at high pH and characterized them morphologically, chemically, and enzymatically. Phase-contrast microscopy revealed the nuclei to be free of cytoplasmic tags and that the nuclear envelopes were isolated as membrane "ghosts." Electron micrographs clearly showed the double-membrane system with nuclear pore complexes which illustrates that the nuclear envelopes were ultra-structurally intact. The nuclear envelopes contained little DNA, low levels of arylesterase or acid phosphatase activity, and undetectable levels of succinate dehydrogenase and 5′-nucleotidase activity. Coomassie blue staining of the nuclear envelope fractions on sodium dodecyl sulfate – polyacrylamide gels for all three cell lines revealed that most of the polypeptides were similar. However, we have identified androgen-dependent peptides of molecular weights 29 000, 32 000, and 34 000 in nuclear envelopes of the androgen-responsive cell line peptide profiles by comparing the nuclear envelopes prepared from the androgen-responsive cell line grown in intact mice, in castrated mice, and in mice which had been injected with testosterone after castration. Further investigation of the androgen regulation of these nuclear envelope peptides may help us understand the molecular mechanisms involved during morphological changes of the nucleus which occur in response to different hormonal environments.
Occurrence of 3-acetamido-3,6-dideoxy-4-O-miethyl-L-glucose in the O-polysaccharide of Vibrio anguillarum
Authors: Banoub Joseph H. , Michon Francis , Cooper Fred
Published: December 2009

The amino sugar 3-acetamido-3,6-dideoxy-4-O-methyl-L-glucose has been characterized in the O-polysaccharide obtained from the bacterial lipopolysaccharide of Vibrio anguillarum. The identity of 1,2,5-tri-O-acetyl-3-acetamido-3,6-dideoxy-4-O-methyl-L-glucitol has been established by electron-impact and chemical-ionization mass spectrometry and by a labeling procedure.
Effect of glutaraldehyde on hemoglobin: functional aspects and Mössbauer parameters
Authors: Guillochon Didier , Vijayalakshmi Mokambes Waran , Thiam-Sow Atsu , Thomas Daniel , Chevalier Alain
Published: December 2009

Glutaraldehyde is widely used for the cross-linking of hemoglobin for blood substitute research or for technological purposes. The effects of this reagent on the biochemical properties of hemoglobin were correlated with Mössbauer data. Human hemoglobin was cross-linked by glutaraldehyde as soluble polymers and insoluble particles. Effects of cross-linking on oxygen affinity, oxidation–reduction potential, autoxidation kinetics, and thermal stability were studied. Stability of cross-linked hemoglobin was specifically studied by Mössbauer spectroscopy. Oxygen affinity is increased, redox potential is decreased, autoxidation rates are increased, and stability towards thermal denaturation is increased. The regeneration of partially denatured hemoglobin by glutaraldehyde cross-linking is shown. Effects of cross-linking on biochemical properties are explained by the hypothesis of the opening of the heme pocket on the distal-histidine side and the concomitant charge transfer from the iron to the oxygen.
Modifications of lipid structure and their influence on mesomorphism in model membranes: the influence of hydrocarbon chains
Authors: Keough K. M. W.
Published: December 2009

The influence of hydrocarbon chains on the temperature (TG–LC) of the gel to liquid-crystalline phase transition of model membranes has been investigated over an extensive variety of phosphatidylcholines (PC). The TG–LC is dependent upon the length of the hydrocarbon chains, on whether or not the chains are saturated or have been modified in some way, and on the position of any modification along the chain. For PC having two different acyl chains (heteroacid PC) in the sn-1 and sn-2 positions, the TG–LC is dependent on the chain position and on the inequivalence of chain penetration into the bilayer. Positional isomers of PC have different TG–LC. The first two double bonds introduced in each chain of a PC cause a much greater reduction in TG–LC and in the enthalpy change of the transition than does the subsequent introduction of additional double bonds. Dipolyunsaturated PC have uncooperative (broad) transitions that occur at low temperatures and have small enthalpy changes. While each PC has unique transitional characteristics, there are a number of patterns in the TG–LC which emerge on consideration of all the available data. One such pattern may be useful in predicting TG–LC from analytical data on the composition and positions of acyl chains of various lipids.
Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions
Authors: Boggs Joan M.
Published: December 2009

The large number of different membrane lipids with various structural modifications and properties and the characteristic lipid composition of different types of membranes suggest that different lipids have specific functions in the membrane. Many of the varying properties of lipids with different polar head groups and in different ionization states can be attributed to the presence of interactive or repulsive forces between the head groups in the bilayer. The interactive forces are hydrogen bonds between hydrogen bond donating groups such as —P—OH, —OH, and —NH3+ and hydrogen bond accepting groups such as —P—O− and —COO−. These interactions increase the lipid phase transition temperature and can account for the tendency of certain lipids to go into the hexagonal phase and the dependence of this tendency on the pH and ionization state of the lipid. The presence or absence of these interactions can also affect the penetration of hydrophobic substances into the bilayer, including hydrophobic residues of membrane proteins. Evidence for this suggestion has been gathered from studies of the myelin basic protein, a water-soluble protein with a number of hydrophobic residues. In this way the lipid composition can affect the conformation and activity of membrane proteins. Since hydrogen-bonding interactions depend on the ionization state of the lipid, they can be altered by changes in the environment which affect the pK of the ionizable groups. The formation of the hexagonal phase or inverted micelles, the conformation and activity of membrane proteins, and other functions mediated by lipids could thus be regulated in this way.
Metabolism and control of lipid structure modification
Authors: Thompson Jr. Guy A.
Published: December 2009

The lipid composition characteristic of a particular cellular membrane can become significantly altered, sometimes quite suddenly, when the cell is placed under environmental stress. In the majority of cases examined, the alterations seem to return the membrane's physical state towards that existing prior to imposition of the stress. The compositional changes are often diverse in their nature and also in their site of origin within the cell. Certain modifications, such as changes in the degree of phospholipid acyl chain unsaturation and in the reordering of fatty acid pairing on specific phospholipids, are now recognized as crucial first responses to stress, while others (e.g., fluctuations in relative proportions of different phospholipid classes and in the sterol:phospholipid ratio) develop more slowly and may represent secondary adjustments to the initial lipid changes. The factors directly responsible for modifying membrane lipid composition are generally unknown at the molecular level, but recent advances provide new clues favoring involvement, in some cases, of the ubiquitous mediator Ca2+. In other cases, the physical state of a membrane may directly modulate the activity of lipid-metabolizing enzymes embedded therein.
Chemical DNA sequencing in the opposite direction to the dideoxy chain termination method, using a single preparation of M13 single-stranded DNA
Authors: Bellemare Guy , Potvin Claude , Simard Claire
Published: December 2009

A strategy has been developed allowing the use of a single preparation of single-stranded DNA clones for chemical DNA sequencing in the opposite direction to the classical dideoxy chain termination method. Oligonucleotide complementary to the 5′-end of the multipurpose cloning sequence, with the proper restriction enzyme, is used to cleave specifically the molecules to expose a unique 5′-end, upstream to the inserted DNA, for the kinase labeling reaction. No further treatments are necessary before Maxam–Gilbert chemical sequencing reactions.
A simple immunological detection method for the direct screening of genes from clone banks
Authors: Lo Reggie Y. C. , Cameron Lynne A.
Published: December 2009

A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.
Metabolism of lysopolyphosphoinositides by rat brain and liver microsomes
Authors: Palmer Frederic B. St. C.
Published: December 2009

Lysophosphatidylinositol 4,5-bisphosphate has been reported to form ion-conducting channels in artificial membranes. If formed in vivo, mechanisms for its removal from cellular membranes would be required. Thus, possible pathways were explored in rat brain and liver microsomes. Since neither lysophosphatidylinositol 4-phosphate nor lysophosphatidylinositol 4,5-bisphosphate were acylated in experiments with [3H]arachidonic acid or [14C]oleoyl CoA, polyphosphoinositides do not participate directly in a deacylation–reacylation cycle as proposed for the postsynthesis enrichment of phosphatidylinositol with arachidonic acid. Similar enrichment in polyphosphoinositides can occur only via the rapid phosphorylation–dephosphorylation cycle linking all three phosphoinositides. Lysophosphatidyl[2-3H]inositol 4,5-bisphosphate and lysophosphatidyl[2-3H]inositol 4-phosphate were rapidly dephosphorylated to 1-acyl-sn-glycero(3)phospho(1)-D-myo-inositol by microsomes from both tissues. Appearance of only trace quantities of radioactive lysophosphatidylinositol monophosphate during the catabolism of lysophosphatidyl[2-3H]inositol 4,5-bisphosphate indicated that the second dephosphorylation step, which was cation independent, was at least as fast as the first step which required Mg2+. In the presence of ATP, CoA, and arachidonic acid, the lysophosphatidylinositol was converted to phosphatidylinositol. This acylation reaction was rate limiting in brain microsomes. Dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate was rate limiting in liver microsomes. Neither the lysopolyphosphoinositides nor the lysophosphatidylinositol produced from them in the reactions were degraded by acyl hydrolases or phosphodiesterases in microsomes from either tissue. Therefore, any lysopolyphosphoinositide formed in vivo would probably be removed by dephosphorylation and recycled to phosphatidylinositol.
Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins
Authors: Gaudreau A. , Ménard L. , Murcia G. de , Poirier G. G.
Published: December 2009

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (≥ 10 μM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA–enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.
A synthesis of N6, N6, N6 -trimethyl-L-lysine dioxalate in gram amounts
Authors: Chen Francis M. F. , Benoiton N. Leo
Published: December 2009

A procedure is described for the synthesis of crystalline N6, N6, N6 -trimethyl-L-lysine dioxalate in gram amounts starting from the commercially available N2-tert-butoxycarbonyl-N6-benzyloxycarbonyl-L-lysine, which is reacted with methyl iodide in methanol in the presence of potassium hydrogen carbonate after deprotection of the side-chain amino group by catalytic hydrogenation. The work-up involves only filtrations and evaporations.
Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehyrogenase multienzyme complex of Escherichia coli
Authors: Adamson S. Robert , Holmes Charles F. B. , Stevenson Kenneth J.
Published: December 2009

The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptable to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the —AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH— group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.
Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5′ regulatory sequences
Authors: Marashi Farhad , Helms Sherron , Shiels Alan , Silverstein Saul , Greenspan Daniel S. , Stein Gary , Stein Janet
Published: December 2009

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5′ and 100 nucleotides of 3′ flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5′ flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5′ flanking sequences of this human H3 histone gene and either human β-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a β-globin mRNA transcript was initiated at the appropriate H3 histone cap site, and this same H3 histone promoter supported expression of a functional CAT protein. The SV40 72 base pair (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of β-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5′ flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.
Participation of the microsomal CDP-diglycerides in the mitochondrial biosynthesis of phosphatidylglycerol
Authors: Stuhne-Sekalec Lidija , Chudzik Jerzy , Stanacev Nikola Z.
Published: December 2009

Participation of microsomal CDP-diglycerides in mitochondrial biosynthesis of phosphatidylglycerol was studied by [3H]palmitoyl, [14C]linoleoyl, and [14C]arachidonoyl CDP-diglycerides and [3H]CDP-diglycerides which were bound to microsomal membranes, incubated with unlabelled mitochondrial membranes, and further incubated in the presence of radioactive sn-glycero-3-phosphate under conditions required for mitochondrial phosphatidylglycerol biosynthesis. Ten to 15% of microsomal radioactive CDP-diglycerides was transferred to mitochondrial membranes and incorporated into mitochondrial radioactive lipids identified as phosphatidylglycerol, phosphatidylglycerophosphate, and, when [14C]linoleoyl CDP-diglycerides were used, diphosphatidylglycerol (cardiolipin).
Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II
Authors: Kashem Mohammed A. , Dunford H. Brian
Published: December 2009

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 °C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I–NADH reaction can be explained in terms of a single ionization of pKa = 4.7 ± 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-H–NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 ± 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II–NADH reaction was observed. Over the pH range of 4–10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 × 105 to5.6 × 102 M−1 s−1 and of HRP-II with NADH varied from 4.4 × 104 to 4.1 M−1 s−1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.
Changes in gene expression during spherulation in Physarum polycephalum
Authors: Bernier François , Seligy Vern L. , Pallotta Dominick , Lemieux Gérald
Published: December 2009

Variation of gene expression during spherulation of Physarum polycephalum microplasmodia has been studied by two-dimensional gel electrophoresis of proteins synthesized in vivo and in vitro. The electrophoretic pattern of total cellular proteins has also been analyzed during the course of differentiation. In all cases, major variations were observed and correlated with the formation of biologically mature spherules. After 24 h of differentiation, numerous specific proteins started to accumulate. After 48 h, a time at which viable spherule formation was complete, the synthesis of a set of late proteins was observed. It has also been shown that the synthesis of actin, the most abundant protein in this organism, was significantly repressed during the period at which the cells accumulated the spherule-specific proteins. The results showed that the spherulation of Physarum involves complex and major changes of protein synthesis, which occur in a temporal sequence. These variations are also observed in the population of translatable mRNA, thus suggesting an important role for RNA transcription.
Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase
Authors: Sadana Jai C. , Khan Bashir M. , Fry Ian V. , Cammack Richard
Published: December 2009

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.
Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver
Authors: Bhavnani Bhagu R. , Woolever C. Allan , Pan Chee Chung
Published: December 2009

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic–pituitary–adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system. These results further suggest that maternal and (or) placental hormones may play an important role in the deposition and utilization of fetal lung and heart glycogen.
Analysis of the Heymann nephritogenic glycoprotein in rat, mouse, and human kidney
Authors: Goodyer P. R. , Mills M. , Kaplan B. S.
Published: December 2009

Passive Heymann nephritis is induced in rats by intravenous administration of antiserum raised against antigens of the renal proximal tubule. Evidence by Kerjaschki and Farquhar indicates that the critical nephritogenic is a high molecular weight glycoprotein (HMWgp) of rat renal brush border membrane. Their immunocytochemical studies also localize the nephritogenic antigen to the glomerular epithelial cell surface and may explain in situ formation of immune complexes at this locus in Heymann nephritis. We have confirmed the observations of Kerjaschki and Farquhar by demonstrating the HMWgp in extracts of rat brush border membrane and isolated glomeruli on sodium dodecyl sulphate – polyacrylamide (SDS–PA) (5%) gels. An antiserum raised to purified rat HMWgp identifies the antigen from rat or mouse kidney on Western blots. However, unlike rodent kidney, we were unable to detect a comparable HMWgp in extracts of human kidney on SDS–PA gels and found no cross-reactive material on Western blots of human brush border membrane proteins. Our observations suggest that human kidney lacks the nephritogenic antigen critical to initiation of Heymann nephritis in rodents.
DNA components of Tipula iridescent virus
Authors: Tajbakhsh S. , Dove M. J. , Lee P. E. , Seligy V. L.
Published: December 2009

Purified Tipula iridescent virus (TIV) from infected Galleria mellonella larvae was shown to consist of four DNA components: L (> 150 kilobase pairs (kbp)), S1 (10.8 kbp), S2 (~6 kbp), and S3 (~3.5 kbp). Analysis of the DNA from sucrose gradient fractionated TIV revealed that the fractions corresponding to partially filled virions have less of L relative to S1. All S components are related to L. S1 and S2 are the most similar, while S3 is a subset of S2. Three recombinant plasmids were isolated from a TIV DNA bank constructed in the BamHI site of pBR322. Two plasmids, pTB19-76 and pTB18-110, are related to S1 and S2 and they contain viral sequences that are repeated in the TIV genome. The third plasmid pTBR1-10 is related to S3. At least part of the variation in TIV genome size (174–246 kbp) obtained by summation of restriction endonuclease derived DNA fragments can be accounted for by the presence of the S components and sequence repetition.
The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide
Authors: Holmes Charles F. B. , Stevenson Kenneth J.
Published: December 2009

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radio-labelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as[Formula: see text]This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH214CONHPhAsO is residue-444 and further establishes its active site role.
Purification and characterization of benzoyl-L-tyrosine ethyl ester hydrolase from the yolk sac membrane of chicken egg
Authors: Sugimoto Yasushi , Yamada Masahiro
Published: December 2009

An enzyme which hydrolyzes benzoyl-L-tyrosine ethyl ester (BTEE) was purified from yolk sac membranes of day-18 chick embryos. The purified BTEE hydrolase has a molecular weight of 110 000, being composed of 70 000 and 40 000 subunits, and preferred synthetic substrates for chymotrypsin to those for trypsin. The optimum pH and temperature of this enzyme were 6.5–7.0 and 40 °C, respectively. The Km value for BTEE of the enzyme was 16 mM at pH 6.5 and 30 °C. The enzyme was inhibited markedly by some chymotrypsin inhibitors but scarcely inhibited by trypsin inhibitors. Magnesium ion acted as potent activator, depending on the enzyme purity and its concentration, whereas p-chloromercuribenzoate and zinc ion inactivated the activity markedly. The BTEE hydrolase was found to hydrolyze proteins such as casein and hemoglobin. These data indicated that the enzyme is a proteinase similar to chymotrypsin. This proteinase could act on yolk proteins, suggesting that it plays an important role in the metabolism of yolk at the yolk sac membrane layer.
Rapid automated synthesis via diisopropyl phosphoramidite in situ activation. Chemical synthesis and cloning of a calmodulin gene
Authors: Alvarado-Urbina G. , Chiarello R. , Roberts D. , Vilain G. , Jurik F. , Christensen L. , Carmona C. , Fang L. , Watterson M. , Crea R.
Published: December 2009

A gene coding for a calmodulin was synthesized and cloned. The chemical synthesis of the gene, coding for 149 amino acids, was achieved by the enzymatic ligation of 61 chemically synthesized DNA fragments. The DNA fragments were synthesized using a solid support with a diisopropyl phosphoramidite intermediate and in situ activation. The automated standard cycle time was 10 min/addition. The synthesizer was designed and constructed from inexpensive, readily available parts and controlled by a Commodore 64 computer. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis.
Regulation of rat liver phosphatidylethanolamine N-methyltransferase by cytosolic factors. Examination of a role for reversible protein phosphorylation
Authors: Pelech Steven L. , Ozen Nilay , Audubert François , Vance Dennis E.
Published: December 2009

The nature of cytosolic factors which modulate the activity of rat liver phosphatidylethanolamine (PE) methyltransferase was investigated. The combined additions of cytosol, Mg∙ATP, and NaF to incubations with rat liver microsomes produced a 1.6-fold activation of the methyltransferase at pH 9.2 and a 1.3-fold stimulation at pH 7.0. Nonhydrolyzable 5′ -adenylylimidodiphosphate could not substitute for ATP, although GTP could. The activation was time dependent, stable to reisolation of the microsomes by ultracentrifugation, and partially preventable by other cytosolic components. Despite these indications that PE methyltransferase might be a substrate for cytosolic protein kinases, cAMP and Ca2+–calmodulin exerted little influence on the activation reaction. Furthermore, microsomal PE methyltransferase activity was unaffected by purified preparations of cAMP-dependent protein kinase, calmodulin–dependent protein kinase, and casein kinase II, nor was methyltransferase activity influenced by the purified catalytic subunits of protein phosphatases 1 and 2A. Cytosol also contained inhibitors of PE methyltransferase which could overcome the Mg∙ATP∙NaF-mediated activation of the enzyme, but were not affected by the thermostable phosphatase inhibitors 1 and 2. Part of this inhibitory activity (apparent molecular mass of 15 × 103 daltons) was insensitive to trypsin and chymotrypsin, stimulated by Mn2+, and partly inhibited by NaF. Therefore, regulation of methyltransferase by reversible phosphorylation, while still a tenable hypothesis, is apparently more complex than previously proposed.
Interaction of steroids with the nuclear envelope
Authors: Lefebvre Y. A. , Venkatraman J. T. , Golsteyn E. J. , Howell G. M.
Published: December 2009

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90 000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.