bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Involvement of Poly(ADP‐ribose) Polymerase in the Degradation of NAD Caused by γ‐Radiation and N‐Methyl‐N‐Nitrosourea
Authors: SKIDMORE Christopher J. , DAVIES Margaret I. , GOODWIN Patricia M. , HALLDORSSON Haraldur , LEWIS Pamela J. , SHALL Sydney , ZIA'EE Abed‐Ali 
Published: March 2005

Both N‐methylN‐nitrosourea and γ‐radiation lower cellular NAD in mouse leukaemia cells (L‐1210) in a dose‐dependent way. The minimum NAD level is reached 2 h after a brief exposure to N‐methyl‐N‐nitrosourea, but within 15 min of y‐irradiation. The cells remain metabolically active; they are able to recover their control NAD levels and are impermeable to trypan blue.Several inhibitors of poly(ADP‐ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5‐methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N‐methyl‐N‐nitrosourea on cellular NAD level; 200 uM thymidine, 500 uM 5‐methyl‐nicotinamide, 500 αM theophylline and 500 αM theobromine prevent the lowering of cellular NAD by y‐irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffective even at this dose.The activity of poly(ADP‐ribose) polymerase in permeabilized cells immediately after y‐radiation increases with dose up to 12 krad, giving a maximal 3.4‐fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both γ‐radiation and N‐methyl‐N‐nitrosourea. The enzyme activity is maximal when the NAD content is decreasing at the highest rate and has returned to normal levels when it ceases falling.In permeabilized cells we can distinguish poly(ADP‐ribose) polymerase and NAD glycohydrolase activity by their differential response to inhibitors. The polymerase is sensitive to 5‐methylnicotin‐amide, theophylline, theobromine and thymidine; the NAD glycohydrolase is sensitive to 5‐methyl‐nicotinamide and theophylline, but not to theobromine and thymidine.We propose that the decrease in cellular NAD level produced by y‐radiation and by N‐methyl‐N‐nitrosourea is caused by an increased flux through poly(ADP‐ribose) mediated by an increased activity of poly(ADP‐ribose) polymerase. This consequently lowers the cellular NAD level. This hypothesis implies an involvement of (ADP‐ribose)n in the cellular response to cytotoxic drugs.
Isolation of Synaptosomal Plasma Membranes from Cholinergic Nerve Terminals and a Comparison of Their Proteins with Those of Synaptic Vesicles
Authors: STADLER Herbert , TASHIRO Tomoko 
Published: March 2005

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5′‐nucleotidase, Na+, K+‐activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000–33000 are found. A gel‐electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr, 160000, 147000, 42000, 34000 and 25000) was made. Comigration of major bands was detected in one‐dimensional gel electrophoresis the 42000‐Mr components. Upon two‐dimensional gel electrophoresis the 42000‐Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin‐like polypeptides is unlikely.Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.
Hepatic Glutamine Metabolism under the Influence of the Portal Ammonia Concentration in the Perfused Rat Liver
Authors: HÄUSSINER Dieter , SIES Helmut 
Published: March 2005

l‐Glutamine, when added to the portal perfusate in a system of isolated perfused rat liver (non‐recirculating open system) at physiological concentration of 0.6 mM, does not lead to an extra production of urea and ammonia, but it does so when ammonium ions at physiological concentration of 0.3 mM are simultaneously present. This stimulatory effect of ammonium ions on glutaminase activity is readily reversible. The effect of portal ammonium ions is half‐maximal at 0.2–0.3 mM and maximal at about 0.6 mM.
The maximal rate of production of urea‐N plus ammonia from glutamine is exhibited at 10–12 mM glutamine, amounting to about 2.6 μmolxmin−1 xg wet weight−1.
The modulatory effects of ammonium ions on the disposition of glutamine by the liver were demonstrated (a) by the extra release of nitrogen from glutamine, (b) by the release of 14CO2 from [U‐14C]glutamine, and (c) by measurement of glutamine uptake.
The simultaneous operation of glutamine synthetase and glutaminase activities in liver was demonstrated to exist (futile cycle), based on 14CO2 release from [U‐14C]glutamine and the positive glutamine balance across the hepatic and portal veins, and further on the effects of methionine sulfoximine. This inhibitor of glutamine synthetase abolished net glutamine release and left the rate of 14CO2 relcase from [U‐14C]glutamine unaltered. Moreover, there is an extra O2 uptake by the liver upon glutamine addition which is not accounted for by extra urea synthesis; the extra O2 uptake increases with the portal glutamine supply. An estimate of the rate of this futile cycle at 0.6 mM glutamine is given, approx. 0.1 μmol × min−1× g wet wt liver−1.
The stimulatory effect of portal ammonium ions on hepatic glutamine breakdown is discussed in terms of an inter‐organ feed‐forward mechanism, e. g. between small intestine and liver during conditions of increased glutamine supply and, therefore, increased requirement of glutamine removal from the circulation.
Nuclear Poly(A) Polymerase Activities in the Rabbit Uterus
Authors: ORAVA Mauri M. , ISOMAA Veli V. , JÄNNE Olli A. 
Published: March 2005

Poly(A) polymerase activities were measured from intact rabbit uterine nuclei and chromatin after a single dose of progesterone, and following a 5‐day treatment with progesterone alone or in combination with estradiol. A single intravenous dose of progesterone (5 mg/kg) to estrogen‐primed rabbits elicited an early increase in both bound and free nuclear poly(A) polymerase activities, the peak activity (about 40% over the controls) being reached 30–60 min posttreatment. The bound enzyme activity seemed to exhibit a second rise that occurred 12 h after progesterone administration. A 5‐day treatment of estrous rabbits with progesterone (1 mg/kg) brought about a sixfold elevation in the bound poly(A) polymerase and a twofold increase in the free nucleoplasmic enzyme activity. A corresponding activation also occurred in the chromatin‐associated poly(A) polymerase. Concomitant administration of estradiol (50 μg/kg) with progesterone significantly depressed progesterone‐elicited increases in the poly(A) polymerases. Changes in the nuclear RNA polymerase I and II were minor during a long‐term progesterone treatment, while the total chromatin template activity, as measured in a transcription system in vitro using wheat germ RNA polymerase II, was significantly reduced after a 5‐day progesterone administration. When estradiol was given along with progesterone, it counteracted the effect of progesterone on the chromatin template.These results suggest that progesterone exerts in the rabbit uterus posttranscriptional actions on the metabolism and processing of mRNA species. Moreover, the antiprogestational action of estrogens was already noted in the regulation of enzymes responsible for completion of mature mRNA synthesis.
Comparison of Poly(A)‐Containing RNAs in Different Cell Types of the Lower Eukaryote Schizophyllum commune
Authors: ZANTINGE Bert , DONS Hans , WESSELS Joseph G. H. 
Published: March 2005

Poly(A)‐containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number‐average length of poly(A)‐containing RNA in total RNA and in purified poly(A)‐containing RNA was estimated as 1100 nucleotides. Number‐average length of poly(A)‐tracts was 33 nucleotides. 2.5% of total RNA is poly(A)‐containing RNA and probably up to 7.5% are non‐polyadenylated polydisperse RNA sequences.Saturation hybridization of poly(A)‐containing RNA to gap‐translated [3H]DNA resulted in 16% of the reactive single‐copy DNA to become S1 nuclease resistant. It was found that purified poly(A)‐containing RNA represented the entire RNA complexity, i. e. 10000 different RNA sequences in S. commune.RNA sequences isolated from morphologically different mycelia and from fruiting and non‐fruiting mycelia were identical for at least 90%.
Improvements in Immunoprecipitation of Specific Messenger RNA
Authors: PAYVAR Farhang , SCHIMKE Robert T. 
Published: March 2005

We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non‐specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (e) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation.We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.
A Toxin Purified from the Venom of Black Widow Spider Affects the Uptake and Release of Radioactive γ‐Amino Butyrate and N‐Epinephrine from Rat Brain Synaptosomes
Authors: GRASSO Alfonso , SENNI Maria Immacolata 
Published: March 2005

A purified neurotoxin from the venom of black widow spider Latrodectus mactans tredecimguttatus, when tested on synaptosomes by means of a perfusion technique, stimulates the release of previously accumulated radioactive transmitters.Black widow spider toxin has also been shown to be a potent inhibitor of the uptake systems for γ‐aminobutyrate and N‐epinephrine. The release from synaptosomes of radioactive N‐epinephrine and γ‐aminobutyrate was rapidly increased by the presence of a low concentration of toxin (∼ 1 nM). The toxin does not appear to cause either non‐specific membrane damage or the release of a non‐transmitter substance like γ‐amino[14C]isobutyric acid.The venom‐induced release of γ‐aminobutyrate was shown not to be dependent on extracellular calcium concentration provided that sodium ions are present. By contrast, that of N‐epinephrine appeared to be Ca‐dependent. The release of γ‐aminobutyrate induced by the toxin is not modified by the presence in the perfusing fluid of tetrodotoxin, dithioerythritol and strontium ions, or by pretreatment of synaptosomes with diaminobutyrate. The mechanism of toxin‐induced release is different from that of a depolarization‐induced release.By correlating previous electrophysiological observations with the results obtained in this biochemical study it may be suggested that the toxin can be used in studies aimed at the identification of components specific to the presynaptic terminal membrane involved in neurotransmitter release.
Inhibition of DNA Synthesis in Mammalian Cells by Daunomycin
Authors: SCHELLINX Jean A. A. , DIJKWEL Peter A. , WANKA Friedrich 
Published: March 2005

The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine liver cells. At low daunomycin concentrations (1 and 2 μM) the rate of [3H]thymidine incorporation decreased progressively with the duration of exposure to the inhibitor. This was accompanied by a shift of nascent DNA intermediates of replicon size to higher sedimentation values on sucrose gradients, indicating that daunomycin preferentially affects the initiation of replicating units, both in asynchronous and synchronized cells.At high daunomycin concentrations (12 μM) the rate of chain growth was also markedly reduced. This was indicated by a rapid and nearly complete cessation of the [3H]thymidine incorporation and an accumulation of nascent DNA intermediates of low molecular weight. These observations are discussed in relation to a pre‐fork mode of DNA synthesis.
On the Structure of the Teichoic Acid from the Cell Wall of Streptomyces antibioticus 39
Authors: SHASHKOV Alexander S. , ZARETSKAYA Malvina Sh. , YAROTSKY Sergei V. , NAUMOVA Irina B. , CHIZHOV Oleg S. , SHABAROVA Zoye A. 
Published: March 2005

The cell walls of Streptomyces antibioticus 39 contain a glycosylated poly(glycero1 phosphate), in which the repeating monomeric unit is O‐α‐d‐galactopyranosyl‐(3–3)‐O‐2‐acetamido‐2‐deoxy‐β‐d‐galactopyranosyl‐(1–1)‐glycerol monophosphate.The localization of the phosphodiesters linkages between hydroxyl groups at positions 2 and 3 of the adjacent glycerol residues and the structure of the glycoside were established by 13C nuclear‐magnetic‐resonance spectroscopy. The spectral data are also in accordance with the results of the methylation analysis and enzyme degradation of the glycoside.
Premature Appearance of Hepatic Phosphoenolpyruvate Carboxykinase in Fetal Rats, Not Mediated by Adenosine 3′: 5′‐Monophosphate
Authors: MENCHER David , SHOUVAL Daniel , RESHEF Lea 
Published: March 2005

Injection of streptozotocin in utero to fetuses elicited a premature appearance of cytosolic hepatic activity of phosphoenol pyruvate carboxykinase. This was due to a precocious initiation of the synthesis of the enzyme. The streptozotocin‐induced appearance of enzyme activity was not mediated by adenosine 3′:5′‐monophosphate since the concentration of the cyclic nucleotide in the liver was unaffected by the antibiotic, the administration of dibutyryladenosine 3′:5′‐monophosphate to streptozotocin‐treated fetuses elicited an additive increase in enzyme activity, and insulin administration in utero repressed the streptozotocin effect while the effect due to dibutyryladenosine 3′:5′‐monophosphate was not inhibited by simultaneous insulin injection.Streptozotocin treatment also caused a small but consistent retardation of fetal growth and a marked reduction of liver wet weight. Histological analysis of the liver demonstrated a premature loss of some hematopoietic elements, while hepatocytes appeared normal. Hepatic protein synthesis was unaffected by the streptozotocin treatment.Streptozotocin treatment had no effect on fetal renal phosphoenol pyruvate carboxykinase activity or kidney wet weight.
A Method for the Estimation of Esterase Synthesis and Degradation and its Application to Evaluate the Influence of Insulin and Glucagon
Authors: HEYMANN Eberhard , MENTLEIN Rolf , SCHMALZ Rainer , SCHWABE Christian , WAGENMANN Frank 
Published: March 2005

The irreversible reaction between liver esterases and the active‐site‐directed inhibitor bis(4‐nitrophenyl)phosphate can be used in vivo both for the estimation of the esterase contents and for the measurement of the esterase degradation rates. A method based on this reaction is described which allows the simultaneous estimation of the rate constants of degradation and synthesis of esterases during a period of change in protein concentration. Rat liver was found to contain about 1 mg of organophosphate‐binding esterases per g of fresh tissue while the microsomal fraction contains about 30 mg of esterases per g of microsomal protein. Esterase degradation and de novo synthesis were shown to remain in equilibrium for a period of at least five days following the injection of 10 mg bis(4‐nitro‐[14C]phenyl)phosphate per kg. The decrease of the relative amount of labeled esterases with time was found to follow first‐order kinetics yielding an average esterase degrading constant of 0.0165 h−1 which corresponds to a half‐life of 42 h. These data were confirmed by an independent experiment using one of the standard procedures for the estimation of degradation rates: [14C]leucine was incorporated and one of the esterases was subsequently isolated by immunoprecipitation. Using isoelectric focussing and dodecyl sulfate electrophoretic methods, the various esterase isoenzymes appeared to have very similar, if not identical turnover rates.This method for the estimation of the turnover characteristics was applied to evaluate hormone effects on liver esterases. The time course of the contents and the turnover of liver esterases was measured under the influence of glucagon treatment in diabetic rats and under the influence of high doses of insulin. The esterase content decreased faster than the average content of microsomal protein under the influence of glucagon. The reverse effect was observed with insulin‐treated rats. Both insulin and glucagon apparently reduced the intracellular esterase turnover in rat liver. Kinetic analysis of the results revealed that insulin mainly lowered the esterase degradation rate, though the rate of esterase synthesis might also have been restricted. In the glucagon‐treated rats the de novo synthesis of esterases was strongly reduced.
Mechanism‐Controlled Stereospecificity
Authors: POLGÁR László , FEJES Judit 
Published: March 2005

The activation parameters of acylation of subtilisin with alkyl and p‐nitrophenyl esters of N‐acylamino acid enantiomers were determined. It was found that (1) the activation entropy is much higher with the nitrophenyl esters than with the corresponding methyl esters, (2) the difference in rate constants between enantiomers is 104–105 with methyl esters whereas it is only of the order of 10 with nitrophenyl esters.The results indicate that the catalytic mechanism is simpler for nitrophenyl esters than for alkyl esters. The simple mechanism requires only general base catalysis, and thus permits more freedom of motion in the transition state, whereas the complex mechanism involves both general base and general acid catalysis. Furthermore, the strikingly low enantiomeric specificity with nitrophenyl esters indicates that not only binding but also the catalytic mechanism is an important factor in determining the stereospecificity of an enzyme.The activation parameters for enantiomeric nitrophenyl ester reactions suggest that structurally related substrates can be transformed by the enzyme in different conformations which may be energetically similar or not. The energetically different conformations may account for the activation enthalpy‐entropy compensation.
Transport of d‐Lactate in Perfused Rat Liver
Authors: SCHWAB Andreas J. , BRACHT Adelar , SCHOLZ Roland 
Published: March 2005

The transport of d‐lactate across the plasma membrane was investigated in hemoglobin‐free perfused rat livers, applying the multiple‐indicator dilution technique (pulse labelling of d‐lactate and indicator substances).The following results were obtained:

The steady state exchange rate at 1 mM d‐lactate was 2.5 μmol × min−1× g wet wt−1. It was proportional to the extracellular concentration in the range between 0.1 and 70 mM.
The transport of d‐lactate was inhibited by l‐lactate and pyruvate; 50% inhibition was observed at 40 mM l‐lactate or 5 mM pyruvate.
The transport was also inhibited by α‐cyanocinnamate and 4,4′‐diisocyanostilbene‐2,2′‐disulfonic acid. The inhibition by cyanocinnamate was complete (with 25 mM) and fully reversible, whereas the inhibition by diisothiocyanostilbenedisulfonic acid was incomplete and irreversible; it was dependent upon the amount of diisothiocyanostilbenedisulfonic acid bound by the liver. Maximal inhibition (80%) was observed with 2 μmol diisothiocyanostilbenedisulfonic acid bound per g wet weight.
The intracellular concentration (ci) of d‐lactate was proportional to the extracellular concentration (ce); the ratio ci/ce was 0.5 throughout the concentration range studied. It decreased in the presence of l‐lactate or pyruvate.

It is concluded that the transport of d‐lactate is carrier‐mediated, and, at least partially, electro‐neutral.
Dissociation of Thyroglobulin Iodination and Hormone Synthesis Catalyzed by Peroxidases
Authors: VIRION Alain , POMMIER Jacques , NUNEZ Jacques 
Published: March 2005

The properties of thyroid peroxidase in the catalysis of thyroglobulin iodination and thyroid hormone synthesis (coupling reaction) were compared to those of horseradish peroxidase. The following results were obtained.

Whereas the thyroid enzyme catalyzes both reactions at the same pH (∼ 7.4) horseradish peroxidase has a pH optimum for iodination of 5.2 and a complex pH dependency for coupling: two peaks of activity were found at pH ∼ 7.5 and ∼ 8.5 with no activity left below pH 5 and above pH 10. These properties of horseradish peroxidase allow a clear dissociation of the iodination and the coupling reactions.
Different experiments were performed in which iodination but not coupling could be obtained with either enzyme; a second incubation of these samples at the same or a different pH and with the same or the other enzyme resulted in different levels of coupling. This procedure revealed the following observations. (a) The low efficiency of horseradish peroxidase in performing the coupling reaction at a low pH is not due to the fact that fewer iodotyrosine residues, potentially able to couple, are formed at this pH. Actually 4 hormonogenic iodotyrosine residues were formed at pH 5.2 with horseradish peroxidase but only two of them were able to couple at this pH. Raising the pH to a more alkaline range resulted in the coupling of these preformed hormonogenic residues, (b) Higher levels of coupling, however, were obtained with horseradish peroxidase only at higher levels of iodination than with thyroid peroxidase suggesting a differential sequence in the iodination by the two enzymes of the different tyrosine residues of thyroglobulin.
The coupling reaction was catalyzed by horseradish peroxidase in the absence of iodide whereas the iodide was required for the thyroid enzyme.