bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the histones from fish erythrocytes
Authors: Miki Brian L. A. , Neelin James M.
Published: December 2009

Because of contentions concerning the generality of cell-specific histone 5 (H5) in nucleated erythrocytes of species other than birds, the erythrocyte histones of carp, trout, perch, black crappie, and white sucker were studied under conditions which minimize proteolysis. All were examined by polyacrylamide gel electrophoresis, and histone 1 (H1) and H5 were purified by cation-exchange chromatography on Amberlite CG-50 and by exclusion chromatography through BioGel P60.A protein homologous to H5 could be isolated and identified from the mature erythrocytes of carp, trout, perch, and black crappie but not from white sucker. The relative amounts of H5 differed extensively and inversely in proportion to H1, implying some functional homology between these proteins. The extremely variable levels of H5, including its apparent absence from one species, suggests that typical H5 is not essential to the function or development of nucleated erythrocytes.Although H1 is the most divergent histone fraction, H5 is also highly variable. Fish erythrocyte H5 differs from avian H5 in relative electrophoretic mobility, ease of elution from Amberlite CG-50 cation-exchange columns, and amino acid composition. H5's from fish tend to have more threonine but less serine, arginine, glutamic acid, and histidine than avian H5's. Fish H5's are more diverse than avian H5's and resemble the H5 homologues from other extremely diverse species; thus avian H5 may be an extreme in specialization of an H1 subfraction.
Fructose-induced adenine nucleotide catabolism in isolated rat hepatocytes
Authors: Smith Camilla M. , Rovamo Liisa M. , Raivio Kari O.
Published: December 2009

The mechanism of fructose-induced nucleotide catabolism was studied using isolated rat hepatocytes in which the adenine nucleotide pool was prelabelled with [14C]adenine. Incubation of these cells with fructose caused a rapid depletion of the adenine nucleotides and a corresponding increase in allantoin. There was no accumulation of radioactivity in adenosine in the presence or absence of the adenosine deaminase inhibitor 9-erythro-(2-hydroxy-3-nonyl)adenine. This confirms the previous hypothesis that fructose-induced adenine nucleotide catabolism occurs by way of AMP deaminase (AMP amino-hydrolase, EC 3.5.4.6).
Energy transduction in the mitochondrionlike bacterium Paracoccus denitrificans during carbon- or sulphate-limited aerobic growth in continuous culture
Authors: Lawford H. G.
Published: December 2009

Paracoccus denitrificans was grown in carbon-limited aerobic continuous culture (critical dilution rate (Dc) = 0.48 h−1). The molar growth yield for carbon (succinate or malate) was constant at about 60 over a broad dilution range (growth rate) from 0.10 to 0.48 h−1. Measurements of the stoichiometry of proton translocation associated with the oxidation of endogenous substrates yielded a ratio of protons ejected from the cell per atom of oxygen consumed (→H+:O) of 8.55 which decreased to 5.85 in the presence of piericidin A (PA), a specific inhibitor of NADH dehydrogenase (EC 1.6.99.3). With starved cells, the observed →H+:O associated with the oxidation of added succinate in the presence of PA was 5.61. These observed →H+:O's represent an underestimation since no correction was made for proton backflow during the short interval of respiratory activity. Aerobic growth of Pc. denitrificans in the chemostat becomes sulphate limited at entering concentrations of sulphate <300 μM. Neither the maximum specific growth rate (measured at Dc) nor the observed molar growth yield for succinate decreased under sulphate limitation. The NADH oxidase in electron transport particles prepared from sulphate-limited cells was completely inhibited by PA. The stoichiometry of proton translocation associated with malate oxidation was similarly unaffected by sulphate limitation. It is concluded that (a) the respiratory chain of aerobic, heterotrophically grown Pc. denitrificans possesses three sites of energy conservation, including site III, (b) the number of protons ejected during the transfer of one pair of reducing equivalents along a region of the electron transport chain equivalent to a single energy-coupling site is 3, and (c) that sulphate limitation does not lead to a loss of proton translocation associated with the cytochrome-independent region of the respiratory chain.
Ammoniagenesis in kidney cortex mitochondria of the rat: role of the mitochondrial dicarboxylate anion transporter
Authors: Cheema-Dhadli Surinder , Halperin Mitchell L.
Published: December 2009

Since glutamine enters rat kidney mitochondria without exchange for an anion, the exit of its carbon skeleton must involve the dicarboxylate anion transporter (malate – inorganic phosphate) for ammoniagenesis to proceed. Therefore, this important mitochondrial anion transporter was studied in isolated renal cortex mitochondria. The phosphate concentration required for half-maximal rates of malate exit from renal cortex mitochondria of normal rats was 1.0 mM. This value was not decreased in renal cortex mitochondria from rats with chronic metabolic acidosis. The maximum velocity of the dicarboxylate transporter was not increased in renal cortex mitochondria from these acidotic rats. These kinetic parameters were similar in liver mitochondria. There was no acute activation of the dicarboxylate carrier when the incubation medium pH was lowered. Thus, there is no demonstrable activation of the dicarboxylate anion transporter in kidney cortex mitochondria of the rat with chronic metabolic acidosis. The significance of these results with respect to the regulation of renal ammoniagenesis is discussed.
A study of cell surface constituents restricting Salmonella minnesota R form glycolipid mR595 fixation to normal rat embryo fibroblasts
Authors: Nigam Vijai N. , Lallier Réal , Brailovsky Carlos A.
Published: December 2009

The nature of the cell surface constituent(s) containing sialic acid that block fixation of bacterial glycolipid mR595 to normal rat fibroblasts was explored. Indirect evidence suggests that a trypsin-sensitive, high molecular weight glycoprotein, present in larger amounts on the surface of normal cells (as compared with transformed rat fibroblasts), may be involved as a blocking agent.
A proposed nucleotide sequence for the 5S ribosomal ribonucleic acid of rainbow trout (Salmo gairdneri)
Authors: Roy Kenneth L.
Published: December 2009

Rainbow trout cell cultures have been exposed to 32P-labelled inorganic phosphate and the labelled RNA has been isolated. The 5S ribosomal ribonucleic acid (5S rRNA) was purified by polyacrylamide gel electrophoresis, then digested with RNase T1 or pancreatic RNase. The products of complete digestion were separated and their sequences determined. These analyses have allowed a sequence to be proposed which differs in eight positions from that of mammalian 5S rRNAs.
Phenylalanyl synthetase function in cultured fibroblasts from subjects with progeria
Authors: Goldstein Samuel , Varmuza Susannah L.
Published: December 2009

Since progeria cells contain a diversity of altered proteins, some aspects of phenylalanyl synthetase function were examined in semipurified extracts of cultured skin fibroblasts using mixed rabbit tRNA as acceptor. No significant differences were found in the Km and Vmax for phenylalanine or ATP in progeria cells compared with controls. Initial velocities of both progeria and control synthetases were lower at late passage owing to either reduced enzyme content or reduced catalytic efficiency. Reverse phase 5 chromatography of tRNAs acylated by progeria and control synthetases gave a single peak of labeled phenylalanine tRNA in all cases with no secondary peaks evident. Total activity of phenylalanyl synthetase in progeria cells was similar to that of control cells at early passage while late-passage control cells had lower specific activities of these synthetases per unit protein.
DNA nicking–closing activity from salmon testis
Authors: Eskin B. , Morgan A. R.
Published: December 2009

Salmon testis is a good source of DNA nicking–closing (N–C) enzyme, as expected for rapidly proliferating cells. Partial purification was obtained but it resulted in an unstable form of N–C enzyme. Cruder fractions are useful for relaxing supercoiled DNA under conditions where other N–C enzymes are inactive. The enzyme is very tolerant of salt concentration, with activity still detectable at 0.6 M NaCl. It is also active at low temperatures with reasonable activity at 0 °C. However, at temperatures greater than 25 °C it is rapidly inactivated. Otherwise, its properties are similar to other eucaryotic N–C enzymes.
Apparent absence of symmetrical transcription in Tetrahymena mitochondria
Authors: Young Paul G.
Published: December 2009

Transcription in Tetrahymena pyriformis mitochondria has been examined for whether or not symmetrical transcription occurs. A ribonuclease A digestion in high-ionic strength was used as a test for the presence of double-stranded RNA (dsRNA) either before or after self-annealing- This test was applied to RNA prepared using pulse lengths of from 1 to 30 min. If symmetrical transcription occurred, then the amount of dsRNA should have varied inversely with pulse length. There was no evidence for the presence of dsRNA other than a ribonuclease-resistant core. A search was also made among the largest transcripts present (> 26S) since this region of a sucrose gradient is enriched in symmetrical transcripts in HeLa cells. Again none were found. In order to allow for possible rapid processing as the reason for the failure to find dsRNA, total cellular RNA was extracted as quickly as possible, and the ribonuclease digest analyzed by Sephadex chromatography. No large pieces of dsRNA were found.
Sterol ordering effects and permeability regulation in phosphatidylcholine bilayers. A comparison of ESR spin-probe data from oriented multilamellae and dispersions
Authors: Butler Keith W. , Smith Ian C. P.
Published: December 2009

The effects of a series of sterols on molecular order and motion in bilayers formed from egg lecithin and dicetyiphosphate were examined and correlations between order and data on permeability to 22Na+ were sought. Electron spin resonance spectra were observed for probes intercalated both in multilamellar dispersions where the effects of motion and orientation are difficult to separate, and in planar multibilayers where the degree of molecular order may be measured even in the presence of slow probe motion. It was concluded from the planar multibilayer data that sterols which increase the degree of ordering of lipid molecules decrease 22Na+ permeability, and that sterols which have the opposite effect on order increase permeability. All the sterols tested lead to decreased rates of motion of the probes. This effect obscures the correlation between order and permeability using data from dispersions.
Technique rapide de preparation des acides ribonucleiques thyroidiens sans phenol
Authors: Besson J.-E. , Champier J. , Chambosse A.-M. , Radisson J. , Berger M.
Published: December 2009

Thyroid tissue was homogenized in 2 M LiCl. The homogenate was allowed to stand 1 h 30 min at 2 °C and then centrifuged. The pellet was suspended in 5% triisopropylnaphthalene sulfonic acid (the sodium salt), 0.05 M Tris–HCl, and 0.1 M NaCl (pH 8). After stirring and centrifuging, the supernatant containing the crude RNA was purified by filtration on Ultrogel AcA 22 (LKB, Sweden). Before adding the sample of crude RNA to the column, pronase was placed on the column. When pronase had entered the gel, we added the sample. The first peak contained pure RNA plus some DNA. The former was precipitated with 2 M LiCl. The RNA species obtained by this technique were undegraded and the yield was 30% better than that of the phenol technique.
Synthesis of and allergic contact dermatitis to bicyclo-[2,2,1]-heptyl-α-methylene-γ-butyrolactones derived from norbornene and camphene
Authors: Schlewer Gilbert , Stampf Jean-Luc , Benezra Claude
Published: December 2009

Three new lactones, 2-oxo-3-methylene-4,7-methanobenzofuran and two α-methylene spirolactones, 3′,3′-dimethylspiro(2-oxo-3-methylenefuran-5,2′-norbornane), were synthesized and their skin activity was tested on one human volunteer and on guinea pigs. The man, sensitized to Frullania, was found sensitive to the above lactones and also reacted to frullanolide and alantolactone. The guinea pigs, experimentally sensitized to alantolactone, cross-reacted to 2-oxo-3-methylene-4,7-methanobenzofuran and the two α-methylene spirolactones, and also to isolantolactone and frullanolide.
The levels of apolipoprotein-E in hypercholesteroiemic rat serum
Authors: Wong Laurence , Rubinstein David
Published: December 2009

The levels of apolipoprotein-E (apo-E) in serum and isolated lipoproteins from diet-induced hypercholesterolemic, and to some extent, hypertriglycerdemic rats were measured by electroimmunoassay. The hypocholesterolemia was accompanied by a mild hypertriglyceridemia. The apo-E was increased by 60% in the hypercholesterolemic serum with a 5- and 50-fold increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) respectively. However, the proportion of apo-E in nascent VLDL isolated from the hepatic Golgi apparatus of hypercholesterolemic rats was significantly decreased. In control serum, 40–50% of the apo-E is found in the density >1.21 g/ml fraction, although this is at least partially due to ultracentrifugation. The aproprotein is absent from the density >1.21 g/ml fraction from hypercholesterolemic serum, suggesting that it is bound more firmly to the lipoprotein complex. It is concluded that the large increases in apo-E in the VLDL and LDL density ranges of serum from hypercholesterolemic rats may in part be accounted for by the utilization of apo-E normally found at higher densities.
Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells
Authors: Sparkuhl Joachim , Setterfield George
Published: December 2009

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, only 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.
Lactoperoxidase-catalyzed oxidation of [4-14C]estradiol
Authors: Jellinck P. H. , Cleveland Shirley
Published: December 2009

The conversion of [4-14C]estradiol to water-soluble products by lactoperoxidase (EC 1.11.1.7) in the presence of added or generated H2O2 was studied using albumin or tyrosine as acceptor. The enzyme was able to catalyze the oxidation and binding of estradiol to albumin even in the absence of 2,4-dichlorophenol at very low concentrations of hydrogen peroxide. Other systems in which H2O2 was replaced by oxygen and Mn2+, light-sensitized riboflavin or glutathione was also shown to be active in the conversion of estradiol to water-soluble products and the effect of inhibitors on these reactions was investigated. Possible mechanisms for the peroxidase-catalyzed formation of these estradiol metabolites are discussed.
Recharacterization of fungal dinucleoside polyphosphate (HS3)
Authors: McNaughton David R. , Klassen Glen R. , Loewen Peter C. , LéJohn Herb B.
Published: December 2009

Three polyphosphorylated dinucleosides given the pseudonyms of HS3, HS2, and HS1 that were erroneously described as diguanosine polyphosphates (LéJohn, H. B., Cameron, L. E., McNaughton, D. R. &Klassen, G. R. (1975) Biochem. Biophys. Res. Commun. 66, 460–467) have been repurified and partially recharacterized. They have proved to be extremely complex molecules; chemical (HCl and KOH hydrolysis), physical (ultraviolet-light spectral analysis and ion-exchange chromatography), and enzymic (nucleotide pyrophosphatase and bacterial alkaline phosphatase hydrolysis) studies showed that (i) all three HS compounds are uracil rich and (ii) only HS3 contains a purine nucleoside and glutamate. The partial structure of HS3 was deciphered as a moiety of ADP – sugar X – glutamate (the mode of attachment of glutamate is obscure) that is covalently linked to another moiety composed of UDP, mannitol, and four phosphates. Sugar X had chromatographic characteristics of ribitol, but the chromatographic isolate also contained a ninhydrin-sensitive entity presumed to be an amino group. Sugar X, therefore, may be an amino sugar polyol. Only the general chemical compositions of HS2and HS1 were determined. Each contained two uridines and HS2 had 10 phosphates whereasHS1 had 12.
The localization within plant cells of enzymes involved in arginine biosynthesis
Authors: Shargool P. D. , Steeves Teresa , Weaver M. , Russell M.
Published: December 2009

Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids.Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.
Analysis of spermidine and spermine in rat liver. Effect of hypophysectomy on polyamine concentrations
Authors: Hall Douglas E. , Symonds G. Wayne , Symonds David L. , Brosnan Margaret E.
Published: December 2009

A simple, rapid, reproducible assay for tissue polyamines using a standard unmodified aminoacid analyzer is described. This technique will allow accurate measurement of the polyamines from as little as 10 mg of liver in less than 1 h. We have used this method to establish the optimum conditions for the extraction of polyamines from liver and to study the effects of hypophysectomy on polyamine content of rat liver. Hypophysectomy caused a fall in RNA, protein, putrescine, and spermidine content per liver cell and prevented the normal increase in spermine in rat liver seen with increasing age.
Studies related to antitumor antibiotics. Part XIV. Reactions of mitomycin B with DNA
Authors: Lown J. William , Weir Gordon
Published: December 2009

The reactions of the antitumor antibiotic mitomycin B with DNA were examined using ethidium fluorescence assays. The following three aspects of mitomycin B action have been studied to compare its behavior with that of mitomycin C: (a) interstrand cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. The greater pKa value of 4.3 found for mitomycin B compared with that of mitomycin C, i.e., 3.2, together with the greater pH dependence of DNA alkylation and interstrand cross-linking and the faster and more extensive cross-linking by mitomycin B at low pH in the absence of reduction, support the suggestion that the aziridine moiety is involved in the initial alkylation of DNA. Mitomycin B, reduced in situ with NaBH4, nicks covalenty closed circular (CCC) PM2 DNA rapidly but less efficiently than mitomycin C in a reaction which is suppressed by (i) superoxide dismutase, (ii) catalase, and (iii) free radical traps showing the intermediacy of O−2∙, H2O2, and OH∙. DNA is cleaved by mitomycin B to which it is covalently attached as well as by the free antibiotic. The addition of intercalated ethidium bromide to DNA prior to treatment with reduced mitomycin B inhibits interstrand cross-linking but not strand scission. The reduced aziridine ring-opened mitomycin B (which lacks the 7-NH2 group of mitomycin C) alkylates DNA and thus provides evidence confirming a previous suggestion that the second covalent link to the DNA is formed at position 10 of the antibiotic.
Ammonia detoxification by the rat kidney in vivo
Authors: Vinay Patrick , Lemieux Guy , Gougoux André
Published: December 2009

The time course of changes in the concentration of metabolites in response to a nontoxic load of ammonia was measured in freeze-clamped kidneys of fasted rats. Following a single NH4HCO3 load, a decrease in tissue concentration of 2-oxoglutarate occurs but this change is small and delayed in relation to the peak of blood ammonia concentration. An immediate but transient increment in tissue glutamine also occurs. No close relationship between the mitochondrial free NAD:NADH ratio calculated from the glutamate dehydrogenase and the 3-hydroxybutyrate dehydrogenase systems is seen during alteration of ammonia concentration. In contrast with previously reported observations in the liver under similar circumstances, no increase in tissue or renal venous blood aspartate or alanine concentration occurs. A constant infusion of NH4HCO3 doubles the tissue glutamine concentration and changes net renal extraction of glutamine to net production. The infusion of NH4+ together with a carbon source (malate, lactate) results in similar increase in tissue and renal vein glutamine concentration. No accumulation of aspartate or alanine are seen. In vitro studies on isolated kidney tubules indicate that the net flux through both aspartate aminotransferase and glutamate dehydrogenase is dependent on the concentration of the reactants as expected for systems in near-equilibrium situation. It is concluded that the rat kidney response to an ammonia load differs from that of the liver despite the apparent existence of a similar network of near-equilibrium systems. Such a difference is best explained by renal glutamine synthesis and sequestration of glutamate as glutamine in vivo.
Effect of the basic amino-acid side chain length and the penultimate residue on the hydrolysis of benzoyldipeptides by carboxypeptidase B
Authors: Moore Graham J. , Benoiton N. Leo
Published: December 2009

The kinetic parameters Km and kcat/Km have been determined for the carboxypeptidase B (CPB, EC 3.4.12.3) catalyzed hydrolysis of benzoylglycyl-DL-homolysine and benzoylglycyl-L-homoarginine. Plots of these data and those for Bz-Gly-Orn and Bz-Gly-Arg (Wolff, E. C., Schirmer, E. W. &Folk, J. E. (1962) J. Biol. Chem. 237, 3094–3099) and Bz-Gly-Lys versus the length of the side chain of the basic amino acid indicate that unlike trypsin (EC 3.4.21.4) (Seely, J. H. &Benoiton, N. L. (1970) Can. J. Biochem. 48, 1122–1131) CPB has a higher binding affinity for a guanidino group than for an amino group at the side chain of the substrate C-terminus. On the other hand, CPB is similar to trypsin (ibid) in that the best substrate would have a side chain length between those of lysine and arginine.Studies with Bz-MeGly-Lys and Bz-Ala-Lys showed that the former is very slowly hydrolyzed by CPB but that the latter is a good substrate, with a high affinity for the enzyme, indicative of considerable participation of the Cα-methyl group of alanine in the binding of the substrate to the enzyme.
L-Ascorbic acid and lysosomal acid hydrolase activities of guinea pig liver and brain
Authors: Hoehn Susan K. , Kanfer Julian N.
Published: December 2009

The effects of L-ascorbic acid deficiency on guinea pig hepatic and brain lysosomal hydrolases were examined. In general, hepatic β-N-acetylhexosaminidase, β-D-glucoronidase, α-D-galactosidase, α-D-mannosidase, and acid phosphatase were elevated in scorbutic animals. This appears to be independent of the starved state. Brain β-D-glucoronidase and acid phosphatase followed a similar pattern to that observed with the liver enzymes, but brain β-N-acetylhexosaminidase was not affected by L-ascorbic acid deficiency. Supplementing the in vitro assay system with L-ascorbic acid decreased the activity of hepatic β-N-acetylhexosaminidase somewhat but had no effect on the brain enzyme. Serum total β-N-acetylhexosaminidase was unaffected by dietary treatments although the activity of β-N- acetylhexosaminidase A tended to increase in the scorbutic animals. Subcellular fractions were obtained from the three groups of animals and the recoveries of protein, β-N-acetylhexosaminidase, and glucose-6-phosphatase estimated.
Conformational studies on wheat embryo 5S RNA using nuclease S1 as a probe
Authors: Barber Calvin , Nichols J. L.
Published: December 2009

Ribosomal 5S 32P-labelled RNA was isolated from imbibing wheat embryos and digested with nuclease S1, a single-strand specific nuclease. The products of enzymic digestion were separated by polyacrylamide gel electrophoresis and identified by fingerprint analysis of their RNase T1 digestion products. The results indicate that the most sensitive portion of the molecule, and hence, the region containing the least helical structure, is close to the 5′-terminus. Similarly, the most resistant portion of the molecule is close to, but does not include, the 3′-terminus. These findings are discussed in relation to proposed models for the secondary structure of 5S RNA.
relA Gene control of bacterial glycogen synthesis
Authors: Bridger William A. , Paranchych William
Published: December 2009

Starvation of Escherichia coli K12 for an amino acid results in the stimulation of bacterial glycogen synthesis in cells containing the relA+ gene, but not in cells carrying the relA− allele. Similarly, a large difference in glycogen content is demonstrable between relA+ and relA− cells in stationary phase. It is concluded that guanosine 5′,3′-bis(diphosphate) (ppGpp) or some related relA -dependent metabolite is involved in the regulation of bacterial glycogen synthesis. Detection of significant basal levels of glycogen in a relA− strain of E. coli and in unstarved relA+E. coli indicates that relA control is not absolutely required for glycogen synthesis but serves as a signal for modulation in response to nutrient availability.
Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12-, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28–31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or'pH 5.1' 105 000 × g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acid and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.
Preparation, characterization, and properties of a novel triple-modified derivative of the Ca2+-dependent protein modulator
Authors: Walsh Michael , Stevens Frits C.
Published: December 2009

A novel, chemically modified derivative of the Ca2+-dependent protein modulator of cyclic nucleotide phosphodiesterase was prepared by the sequential treatment of the native protein with tetranitromethane, 1,2-cyclohexanedione, and diethylpyrocarbonate. Three derivatives were isolated during the synthesis, i.e., a single-modified derivative containing two modified tyrosine residues per mole; a double-modified derivative containing two modified tyrosines plus five modified arginine residues per mole; and a triple-modified derivative containing two modified tyrosines, five modified arginines, and one modified histidine residue per mole. Intermolecular cross-linking was observed to occur as a minor side reaction during nitration of the native protein with tetranitromethane. All the derivatives were examined for phosphodiesterase-stimulating activity and troponin C like activities. All derivatives were found to retain the capacity of the native protein to stimulate modulator-deficient phosphodiesterase; furthermore, in each case, the stimulation of phosphodiesterase was a Ca2+-dependent process. On the other hand, both the double- and triple-modified modulators lost the troponin C like activities, i.e., the Ca2+-dependent change in mobility in urea–polyacrylamide gels and the ability to interact with troponin I in the presence of Ca2+ to form a urea-stable complex, while the nitrotyrosyl modulator retained these properties of the native protein.
Preparation of cell populations with stabilized erythropoietic potential from the primitive streak chick blastodisc: Some implications for control of gastrulation
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: December 2009

Improved methods for preparation from primitive streak chick blastodiscs of cell suspensions capable of forming erythroid cells in culture have been developed. When blastodiscs were preincubated with hyaluronidase in the absence of collagenase before cell dispersion and a high concentration of methyl-α-mannoside was present in all media, the yields of cells were some 10-fold higher than those obtained by former procedures. Cell suspensions obtained consisted almost entirely of viable cells, yielded large numbers of free mature erythrocytes in liquid culture, and formed erythroid colonies and bursts in solidified medium. The capacity to form differentiated cells after re sedimentation through Ficoll density gradients was partly stabilized.Addition of egg yolk homogenate to the blastodiscs immediately following treatment with hyaluronidase and to all media used thereafter largely stabilized the capacity to form erythroid cells during re sedimentation through Ficoll density gradients.Possible relevance of observations made during development of the procedures to the control of onset of cell migration in the process of gastrulation is indicated.
Lipid compositional manipulation in Acholeplasma laidlawii B. Effect of exogenous fatty acids on fatty acid composition and cell growth when endogenous fatty acid production is inhibited
Authors: Silvius John R. , McElhaney Ronald N.
Published: December 2009

A variety of potential inhibitors of de novo fatty acid biosynthesis have been tested for activity in Acholeplasma laidlawii B. Two compounds, avidin and N,N-dimethyi-4-oxo-2-trans-dodecenamide (CM-55), an antimicrobial fatty amide, strongly inhibit de novo biosynthesis without nonspecific toxic effects at moderate dosages. Avidin is the more potent inhibitor, abolishing de novo fatty acid synthesis and greatly reducing the chain elongation of exogenous fatty acids at levels of 25 U/ℓ. CM-55 gives complete inhibition of de novo biosynthesis only at low temperatures and inhibits exogenous fatty acid elongation to a variable extent. However, CM-55 is still a more potent antilipogenic agent in this organism than is the fungal antibiotic cerulenin. Cells cultured with avidin grow only when one or more exogenous medium- or long-chain fatty acids are added to the growth medium. The extent of cell growth under these conditions depends primarily on the physical properties of the exogenous fatty acid(s). In general, fatty acids giving diacylglycerolipids of very high or very low fluidity are unsuitable growth substrates, while those whose diacylglycerol derivatives are of intermediate fluidity support fair to good cell growth.
Role of pyridoxine in the metabolism of putrescine in the rat
Authors: Sourkes Theodore L. , Missala Krystyna
Published: December 2009

1. The ability of rats to metabolize radioactive putrescine to 14CO2 in vivo has been studied. 2. Animals made deficient in pyridoxine exhibit a significantly lower rate of catabolism of the diamine. 3. There does not appear to be an important interaction between the effects of the deficiency and those stemming from treatment of the animals with the diamine oxidase inhibitor aminoguanidine. 4. These results favour the concept of a role of pyridoxal cofactor in the metabolism of diamines, presumably at the diamine oxidase stage.
Guanosine triphosphate catabolism in human and rabbit erythrocytes: Role of reductive deamination of guanylate to inosinate
Authors: Henderson J. Frank , Zombor George , Burridge Philip W.
Published: December 2009

The reductive deamination of guanylate to inosinate was demonstrable but occurred at low rates in human and rabbit erythrocytes incubated in vitro with or without glucose. However, the process was considerably accelerated in erythrocytes incubated with deoxyglucose.In human erythrocytes incubated with deoxyglucose, deamination was the major pathway of catabolism of guanylate; little or no guanylate was dephosphorylated. In rabbit erythrocytes, guanylate was both deaminated and dephosphorylated. Inosinate formed from guanylate was metabolized only by dephosphorylation in human erythrocytes, but in rabbit erythrocytes, it was also converted to xanthylate.
A study of the localization of high mobility group proteins in chromatin
Authors: W. Beatriz Levy , Dixon Gordon H.
Published: December 2009

High mobility group (HMG) proteins from fetal calf thymus and mouse brain chromatin were purified and compared electrophoretically. The four major HMG proteins characteristic of fetal calf thymus chromatin (HMG's 1, 2, 14, and 17) were also found to be present in mouse brain chromatin.Nuclei from these two eucaryotic tissues were digested with DNase I and micrococcal nuclease and the acid-soluble proteins solubilized by the two nucleases in both tissues were analyzed on starch gels.Limited digestion of fetal calf thymus nuclei with DNase I led to the solubilization of a substantial fraction of proteins HMG-1 and HMG-2 together with smaller amounts of H1. In addition, limited digestion with micrococcal nuclease released approximately 70% of HMG's 1 and 2 and variable amounts of H1 into the soluble fraction. The observation that HMG proteins 1 and 2 are selectively solubilized under conditions in which active genes have been shown to be preferentially digested in various other cell types suggests their selective association with chromatin regions which are transcriptionally competent.
The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the ββ′ subunits of RNA polymerase following a nutritional shiftup of Escherichia coli
Authors: Boyle Stephen M. , Chu Frederick , Brot Nathan , Sells Bruce H.
Published: December 2009

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the β and β′ subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT'−). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the β and β′ subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT− strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and β and β′ subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the β and β′ subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT− strain versus the timing when the new rates of stable RNA, ribosomal protein, and β and β′ subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.
Binding of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli to phospholipid vesicles
Authors: Bragg P. D. , Hou C.
Published: December 2009

Incubation of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli with phospholipid vesicles resulted in binding of the enzyme to the lipid. Binding was observed with vesicles of soybean phospholipid (asolectin), phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and cardiolipin. Binding was not affected by alterations in pH in the range of pH 6.5 to 8.5, by ionic strength, or by the presence of Mg2+. Loss of the δ subunit from the enzyme had no effect on binding. However, removal of the δ and ε subunits by treatment of the enzyme with trypsin prevented binding to phospholipid. This treatment also removed a small portion (<2000 daltons) of the α subunit. It is concluded that the ATPase of E. coli binds to phospholipid vesicles mainly by nonpolar interactions through the α and (or) ε subunits of the enzyme.
The topological trapping of circular DNAs on agarose: Unexpected restrictions on DNA rotation
Authors: Lee Jeremy S. , Morgan A. Richard
Published: December 2009

DNA linked to an insoluble matrix has many potential applications. In some cases, it is highly desirable that the DNA be chemically unaltered, and for this reason, we have developed methods for topologically trapping circular DNAs on agarose. Open circular (oc) DNA containing at least one nick is readily trapped on agarose which has been heated or dissolved in sodium perchlorate to destroy secondary structure and then gelled by cooling or dialysis respectively. On the other hand, covalently closed circular (ccc) DNA of superhelix density −0.12 (PM2 DNA) is only poorly trapped unless first relaxed by topoisomerases or with the appropriate addition of an intercalating drug. When the oc DNA – agarose was used in a procedure for rapidly obtaining binding constants of drugs to DNA, the binding constant of ethidium was found to be considerably less than that expected. On addition of calf thymus topoisomerase to the binding-assay mixture, the ethidium-binding constant increased to the expected value. Thus, although free oc DNA is topologically unrestricted, oc DNA trapped in agarose must be rotationally constrained such that addition of ethidium introduced supercoils. The nature of these constraints is discussed with respect to the known structure of agarose bihelices.
Differential synthesis of the polypeptides of aldehyde dehydrogenase and NAD(P)H: flavin oxidoreductase in the bioluminescent bacterium Beneckea harveyi
Authors: Bognar Andrew , Michaliszyn George , Meighen Edward
Published: December 2009

The proteins of the bioluminescent bacterium Beneckea harveyi have been labelled with [3H]leucine prior to the induction of bioluminescence, and with [14C]leucine during the development of the bioluminescent system. An aliphatic aldehyde dehydrogenase and a NAD(P)H: flavin oxidoreductase, two enzymes that may be directly involved in the metabolism of the substrates (aldehyde, FMNH2) for the luminescent reaction catalyzed by luciferase, were purified and the isotope ratios of their respective polypeptide chains determined after sodium dodecyl sulfate gel electrophoresis. A comparison of these isotope ratios to (a) the isotope ratios of the induced polypeptide chains of luciferase, purified in the same experiment, and (b) the average isotope ratio for the proteins synthesized in concert with growth has provided direct evidence that the synthesis of aldehyde dehydrogenase but not NAD(P)H: flavin oxidoreductase is induced during the development of bioluminescence.
Cloning and expression of the glycerol-3-phosphate transport genes of Escherichia coli
Authors: Weiner Joel H. , Lohmeier Elke , Schryvers Anthony
Published: December 2009

The two thousand Escherichia coli: Col E1 hybrid plasmid strains of the Clarke and Carbon colony bank (Clarke, L. &Carbon, J. (1976) Cell 9, 91–96) were screened by conjugation for those that correct the deficiency of a mutant unable to transport glycerol-3-phosphate. Six strains harbouring recombinant plasmids carrying the glpT region were identified and characterized with respect to plasmid size and transport properties. The initial rate of glycerol-3-phosphate transport in both whole cells and membrane vesicles prepared from such strains was elevated 3- to 10-fold over strains carrying random DNA inserts, whereas the Km of glycerol-3-phosphate transport was near 12 μM in both experimental and control strains. Four of the six glpT carrying plasmid strains demonstrated elevated levels of the anaerobic glycerol-3-phosphate dehydrogenase coded for by the neighbouring glpA gene.We have transferred the glpT hybrid plasmids into a minicell-producing strain of E. coli X1197 and have used the minicells for specific in vitro synthesis of plasmid-coded proteins. The glpT plasmids code for a 40 000 polypeptide which is localized in the periplasmic space. In addition, they code for a membrane-associated protein of 26 000 which may be the carrier polypeptide.
The defect in transcellular transport of phosphate in the nephron is located in brush-border membranes in X-linked hypophosphatemia (Hyp mouse model)
Authors: Tenenhouse H. S. , Scriver C. R.
Published: December 2009

We purified renal cortex brush-border membranes from mutant hemizygous hypophosphatemic (Hyp/Y) mice and male control (+/Y) littermates. Tenfold purification of mutant and wild-type membranes was obtained. Phosphate enters +/Y brush-border membrane vesicles by a saturable Na+-dependent arsenate-inhibited component and also by a diffusional component observed in the presence of a potassium gradient. Phosphate is not bound or incorporated significantly by mouse brush-border membrane vesicles. Parallel studies with rat renal cortex brush-border membrane vesicles revealed that phosphate and D-glucose transport in rat and mouse vesicles are similar and have the characteristics reported by other workers. Brush-border membrane vesicles prepared from Hyp/Y renal cortex have significant (p < 0.001) partial loss of phosphate transport on the Na+-dependent arsenate-inhibited component. D-Glucose transport is not affected. Our previous studies reveal that other components of transcellular phosphate flux in kidney are normal. Therefore, we conclude that the mutant gene product in the Hyp mouse is confined to the brush-border membrane. Stability of the X-chromosome in mammalian evolution implies that the same gene product is involved in the classic human disease, familial 'vitamin D resistant' X-linked hypophosphatemia.
Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe
Authors: Chan William W.-C. , Enns Caroline A.
Published: December 2009

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has no effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subunit in gel electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.
Transport of RNA from nucleus to cytoplasm following mitogenic stimulation of human lymphocytes
Authors: Mitchell Mary , Bard Enzo , L'Anglais Rod , Kaplan J. G.
Published: December 2009

An improved method affording clean separation of nuclei from cytoplasm was utilized to study the kinetics of appearance of [3H]uridine-labelled polyadenylated (poly(A)+) and nonpolyadenylated (poly(A)−) RNA in the cytoplasm of human lymphocytes stimulated with the mitogen concanavalin A (Con A). While our previous and present studies utilizing lymphocyte preparations purified on Ficoll-Hypaque gradients had shown that the earliest observable increase in labelled total cell RNA occurred after 12 h of exposure to Con A, we were here able to detect highly significant increases in levels of uridine-labelled poly(A)− RNA in the cytoplasm of lymphocytes which had been exposed to mitogen for 6 h; these cells had been pulsed with [3H]uridine for 2 h prior to harvest. This suggested that the early effects of mitogen may be to increase processing and transport of RNA rather than to increase the production of gene transcripts. At least 13 h of exposure to Con A was required before labelled poly(A)+ RNA appeared in the cytoplasmic extracts of stimulated cells and it did not appear in those of resting cells. The lag between addition of isotope and appearance of labelled RNA in cytoplasm was inversely related to the duration of incubation of the cells with mitogen. However, once the label appeared in the cytoplasm, its rate of appearance and its final level were similar at both 12 and 36 h of incubation with Con A. These kinetic data also suggest that mitogen acts mainly to accelerate the processing and transport of RNA at least during the first 12 h of mitogenic stimulation. A concentration (0.05 μg/ml) of actinomycin D which is known to inhibit specifically the transcription of nucleolar RNA in a variety of cell types was found to affect the transcription of poly(A)+ RNA in human lymphocytes; in some experiments, specific inhibition of nucleolar RNA synthesis was observed at 0.005 μg actinomycin D per millilitre.