Fluorescent cytokinins: Stretched-out analogs of N6-benzyladenine and N6-(Δ2-isopentenyl) adenine

We have synthesized and compared the cytokinin activities in the tobacco bioassay of a series of benzologs of 6-(3-methyl-2-butenylamino)purine (N⁶-(Δ²-isopentenyl)adenine) (1a) and 6-benzylaminopurine (N⁶-benzyl-adenine) (1c). The linear benzo analogs 8-(3-methyl-2-butenylamino)imidazo[4,5-g]quinazoline (2b) and 8-benzyla-minoimidazo[4,5-g]quinazoline (2c) are active, while 9-(3-methyl-2-butenylamino)imidazo[4,5-f]quinazoline (3b) and 6-(3-methyl-2-butenylamino)imidazo[4,5-h]quinazoline (4b) are slightly active and 9-benzylaminoimidazo[4,5-f]-quinazoline (3c) and 6-benzylaminoimidazo[4,5-h]quinazoline (4c) are inactive. Compounds 2b and 2c represent the first examples of active cytokinins containing a tri-heterocyclic moiety. The above series of compounds demonstrates structural factors that affect cytokinin activity. These compounds also have interesting fluorescence properties which could render them useful as probes to study the mechanism of cytokinin action.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by α-cyano-4-hydroxycinnamate (Short Communication)

alpha-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.     

Studies on the inhibition of hepatic lipogenesis by N6,O2′-dibutyryl adenosine 3′,5′-monophosphate

Fatty acid synthesis by isolated liver cells is dependent upon the availability of lactate and pyruvate. A lag in fatty acid synthesis is explained by time being required for lactate and pyruvate to accumulate to maximum concentrations in the incubation medium. The initial rate of fatty acid synthesis is not linear with cell concentration, being disproportionately greater at higher cell concentrations because optimal lactate and pyruvate concentrations are established in the medium more rapidly. The accumulation of lactate and pyruvate is inhibited markedly by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. This accounts in part for the inhibition of fatty acid synthesis caused by this cyclic nucleotide. Other sites of action are apparent, however, because exogenous lactate plus pyruvate only partially relieves the inhibition. The profile of metabolic intermediates suggests that N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate inhibits the conversion of glycogen to pyruvate and lactate by decreasing the effectiveness of phosphofructokinase and pyruvate kinase.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ²-Isopentenylpyrophosphate:5′AMP Δ²-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ²-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP^ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn²⁺, Zn²⁺, and Mg²⁺ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mm Zn²⁺, and 25 °C) the K_m values for 5′AMP and Δ²-isopentenylpyrophosphate were 1.0 × 10^?7m and 2.2 × 10^?6m, respectively.     

Induction of lipolysis in vitro and loss of body fat in vivo by zinc-α2-glycoprotein

Loss of adipose tissue in cancer cachexia has been associated with tumour production of a lipid-mobilizing factor (LMF) which has been shown to be homologous with the plasma protein zinc-α₂-glycoprotein (ZAG). The aim of this study was to compare the ability of human ZAG with LMF to stimulate lipolysis in vitro and induce loss of body fat in vivo, and to determine the mechanisms involved. ZAG was purified from human plasma using a combination of Q Sepharose and Superdex 75 chromatography, and was shown to stimulate glycerol release from isolated murine epididymal adipocytes in a dose-dependent manner. The effect was enhanced by the cyclic AMP phosphodiesterase inhibitor Ro20-1724, and attenuated by freeze/thawing and the specific β3-adrenoreceptor antagonist SR59230A. In vivo ZAG caused highly significant, time-dependent, decreases in body weight without a reduction in food and water intake. Body composition analysis showed that loss of body weight could be attributed entirely to the loss of body fat. Loss of adipose tissue may have been due to the lipolytic effect of ZAG coupled with an increase in energy expenditure, since there was a dose-dependent increase in expression of uncoupling protein-1 (UCP-1) in brown adipose tissue. These results suggest that ZAG may be effective in the treatment of obesity.     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

5′-Deoxypyridoxal inhibition of glucocorticoid receptor binding in HeLa S3 cells and rat thymocytes

Recent evidence suggests a possible role for pyridoxal 5′-phosphate, the active physiological form of vitamin B₆, in steroid hormone action (1–3). We now report that 5′-deoxypyridoxal, a synthetic vitamin B₆ antagonist, causes a rapid and complete loss of dexamethasone receptor binding in cytosol preparations in whole HeLa S₃ cells or in rat thymocytes. This effect is concentration and time dependent, and is specific for 5′-deoxypyridoxal. In whole cell incubations of either HeLa S₃ cells or rat thymocytes at 37°C, a 50% reduction in [³H]dexamethasone binding was observed in the presence of 0.25 mM 5′-deoxypyridoxal. Cytotoxicity was not evident in rat thymocytes or HeLa S₃ cells incubated with 3.0mM or 1.0mM 5′-deoxypyridoxal respectively. Pyridoxal 5′-phosphate, 5′-deoxypyridoxine and 5′-deoxypyridoxamine were ineffective in causing a loss of steroid receptor binding. These studies suggest that 5′-deoxypyridoxal may be an effective non-steroidal compound which can effect the binding of glucocorticoids to specific receptor proteins in whole cells.     

Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-ethyl-N′-nitro-N-nitrosoguanidine

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16 h. DNA synthesis peaked 20 h after removal of nutrient stress and declined.Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.     

The inhibition of hexose transport and metabolism by small amounts of adenosine 5′-triphosphate in isolated rat adipocytes

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μm, ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3-O-methylglucose transport. ATP inhibited the V of insulin-stimulated 3-O-methylglucose transport, but had no effect on the K_m. The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μm ATP. However, when hexose transport was blocked with cytochalasin B (50 μm), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.     

The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [¹⁴C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.     

Stimulation by 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver (Short Communication)

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.     

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.     

Influence of caffeine and 3-aminobenzamide in G2 on the frequency of chromosomal aberrations induced by thiotepa,mitomycin C and N-methyl-N-nitro-N′-nitrosoguanidine in human lymphocytes

The effects of post-treatments with caffeine in G₂ on the frequency of chromosomal aberrations induced by thiotepa, mitomycin C and N-methyl-N-nitro-N′-nitrosoguanidine were studied in human lymphocytes. Caffeine was found to potentiate the frequency of chromatid aberrations induced by all 3 S-dependent agents tested; the most striking enhancement being obtained when caffeine was present during the last 1.5 h before harvesting. Post-treatments in G₂ with 3-aminobenzamide had no influence on the aberration frequency induced by thiotepa and N-methyl-N-nitro-N′-nitrosoguanidine.     

The hormonal regulation of enzymes in prenatal and postnatal rat liver. Effects of adenosine 3′,5′-(cyclic)-monophosphate

1. The administration of glucagon, cAMP [adenosine 3',5'-(cyclic)-monophosphate], BcAMP [6-N-2'-O-dibutyryladenosine 3',5'-(cyclic)-monophosphate] or adrenaline to foetal rats during the last 2 days of gestation evoked the appearance of tyrosine aminotransferase and enhanced the accumulation of glucose 6-phosphatase in the liver. In foetuses 1-2 days younger only BcAMP was effective. After birth liver glucose 6-phosphatase no longer responds to glucagon or BcAMP. Tyrosine aminotransferase is still inducible by these agents in 2-day-old rats, but not in 50-day-old rats. After adrenalectomy of adults glucagon or BcAMP can enhance the induction of the enzyme by hydrocortisone. The results indicate that the ability to synthesize tyrosine aminotransferase and glucose 6-phosphatase when exposed to cAMP develops sooner than the ability to respond to glucagon with an increase in the concentration of cAMP; the responsiveness of enzymes to different hormones changes with age. A scheme illustrating the sequential development of competence in regulating the level of an enzyme is presented. 2. Actinomycin inhibited the effects of glucagon and BcAMP on liver tyrosine aminotransferase and glucose 6-phosphatase in foetal rats. Growth hormone, insulin and hydrocortisone did not enhance the formation of these enzymes. 3. The time-course of accumulation of glucose 6-phosphatase in the kidney is different from that in the liver. Hormones that increase the accumulation in foetal liver do not do so in the kidney of the same foetus or in the livers of postnatal rats.     

Depolymerization of heparin with diazomethane. Structure of N,O-methylated,even-numbered oligosaccharides produced by β-eliminative cleavage of the 2-amino-2-deoxyglycosylic linkage

The long-period reaction of heparin with excess diazomethane at 20° resulted in cleavage at the β-position of the uronic acid carboxyl group to give a mixture of methyl α- and β-glycosides of N,O-methylated di-, tetra-, and hexa-saccharides having a 4,5-unsaturated uronic acid, nonreducing end-group. The major disaccharides obtained were methyl O-(4-deoxy-3-O-methyl-α-l-threo-hex-4-enopyranosyluronic acid 2-sulfate)-(1→4)-2-deoxy-3-O-methyl-2-(N-methylsulfoamino)-α- and -β-d-glucopyranoside. The reaction of heparin at 4° yielded a mixture of methylated, higher-molecular-weight oligosaccharides, which retained some affinity for antithrombin III-Sepharose.     

The control of growth of mouse mastocytoma Cells by N6,O2′-dibutyryladenosine cyclic 3′,5′-monophosphate

Summary Addition of N⁶,O^2′-Dibutyryladenosine cyclic 3′,5′ monophosphate (DB cyclic AMP) plus theophylline or transfer to medium containing 0.2% serum slowed the growth of cultured mouse mastocytoma cells and eventually arrested their growth in G1 phase. Examination of the properties of cells arrested by either procedure suggested that the drugs arrested cells in G1 phase 1.5–2 h after the point of low serum arrest. Cycloheximide prevented the recovery of cell growth after low serum or drug-induced arrest demonstrating that protein synthesis was necessary to pass either growth restriction point. Cordycepin also prevented drug-arrested cells from progressing into cycle indicating a requirement for RNA synthesis to overcome the drug-induced growth arrest. Evidence is also presented that DB cyclic AMP prevented the cells receiving a pulse of calcium necessary to proceed past the DB cyclic AMP-sensitive growth restriction point. It is suggested that high cyclic AMP levels prevent mastocytoma cells from receiving a surge of calcium in G1 phase that is necessary if the cells are to proceed to S phase and eventually divide.     

The oxidation of cat,human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α₂^Humanβ₂^Cat and α₂^Catβ₂^Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α₂^Catβ₂^Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α₂^Humanβ₂^Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.