Patterns of adenine metabolism and caffeine biosynthesis in different parts of tea seedlings

Contents of purine alkaloids in different parts of tea ( Camellia sinensis L. cv. Yabukita ) seedlings, seeds and tissue cultures were determined with high-performance liquid chromatography. More than 99% of the caffeine detected was in the leaves of the 4-month-old seedlings. The amount expressed per g fresh weight was higher in older leaves. Theobromine, a precursor of caffeine biosynthesis, was found only in younger leaves. Zero or only trace amounts of theophylline, a degradation product of caffeine, were found in the seedlings. Almost all the caffeine in tea seeds was found in the seed coats. Theobromine and theophilline could not be detected in any part of the seeds.
Tracer experiments using [8-¹⁴C]-adenine indicate that (i) caffeine biosynthesis from [8-¹⁴C]-adenine occurs only in younger leaves,(ii) "salvage" of [8-¹⁴C]-adenine for nucleic acid synthesis takes place in all parts of the seedlings, (iii) considerable degradation of [8-¹⁴C]-adenine by conventional purine degradation pathway via uric acid takes place in roots and lower parts of stem tissue.
The results strongly suggest that caffeine is synthesized in younger leaves and accumulated within the leaves. Both caffeine contents and its synthetic activity from adenine were extremely low in tissue culture of tea.     

Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase)

Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, l-[4.¹⁴C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted l-[4-¹⁴C]aspartic acid is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radiactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthetized enzymatically, using a partial purified preparation of SAICAR synthetase from chicken liver. To the purified [¹⁴C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADPMgCl₂ or buffer alone, and to initiate the reaction, a 12 000 × g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [¹⁴C]SAICAR, l-[4-¹⁴C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40% KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of the Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme.     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Involvement of purine nucleotide synthetic pathways in gonadotropin-induced meiotic maturation in mouse cumulus cell-enclosed oocytes

This study was carried out to test the hypothesis that purine nucleotide-generating pathways are required for ligand-stimulated oocyte maturation in meiotically arrested cumulus cell-enclosed oocytes. Oo-cytes from hormonally primed, immature mice were cultured overnight in Eagle's minimum essential medium containing dibutyryl cyclic AMP (dbcAMP) (to maintain meiotic arrest), plus either mycophenolic acid or alanosine (inhibitors of guanyl and adenyl nucleotide production, respectively). Follicle-stimulating hormone (FSH) was added either at the outset of culture or after a 3-hr preincubation period. Under either of these conditions, the inhibitors suppressed FSH induction of germinal vesicle breakdown (GVB). In addition, the potency of FSH as an inducer of GVB was reduced following the 3-hr preincubation period, but this could be prevented if nucleotide precursors such as hypoxanthine, guanosine, or adenosine were included during the first 3 hr. Furthermore, preincubation had little effect on FSH induction of GVB when hypoxanthine was used to maintain meiotic arrest for the entire culture period. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, could not mimic this protective effect of hypoxanthine. Azaserine and aminopterin, inhibitors of purine de novo synthesis, blocked hormone-triggered maturation in dbcAMP-arrested oocytes, but had little effect on hypoxanthine-arrested oocytes. The effect of azaserine on dbcAMP-treated oocytes could be reversed by the inclusion of AICA riboside, a compound that can be taken up by cells and phosphorylated to form AICAR, which can enter the purine de novo pathway at a point distal to the sites of azaserine inhibition. FSH was stimulatory to purine de novo synthesis, while azaserine, aminopterin, hypoxanthine, and AICA riboside all suppressed de novo synthesis in the presence or absence of FSH, with dbcAMP having no effect. HPLC analysis of ¹⁴C-hypoxanthine metabolism in oocyte-cumulus cell complexes revealed that changes in the pattern of purine metabolism did not mediate the meiosis-inducing effect of FSH. These data support the conclusion that purine nucleotide-generating pathways are vital participants in the mechanism(s) regulating hormone-induced meiotic maturation, and that either the de novo or salvage pathway can fulfill this nucleotide requirement. Mol Reprod Dev 46:155–167, 1997. © 1997 Wiley-Liss, Inc.     

Sequence features of Oxytricha trifallax (class Spirotrichea) macronuclear telomeric and subtelomeric sequences

We sequenced and analyzed the subtelomeric regions of 1356 macronuclear "nanochromosomes" of the spirotrichous ciliate Oxytricha trifallax. We show that the telomeres in this species have a length of 20 nt, with minor deviations; there is no correlation between telomere lengths at the two ends of the molecule. A search for open reading frames revealed that the 3' and 5' untranslated regions are short, with a median length of approximately 130 nt, and that surprisingly there are no detectable differences between sequences upstream and downstream of genes. Our results confirm a previously reported purine bias in the first approximately 80 nucleotides of the subtelomeric regions, but with this larger data set we curiously detected a 10 bp periodicity in the bias; we relate this finding to the possible regulatory and structural functions these regions must serve. Palindromic sequences in opposing subtelomeric regions, although present in most sequences, are not statistically significant.     

Mathematical modelling for the generation of L-[3-2H,3-13C]lactic acid isotopomers by erythrocytes exposed to either D-[1-13C]glucose or D-[6-13C]glucose in the presence of2H2O

Cytosolic purine nucleoside phosphorylase (PNPase) is a well known, and described enzyme which exists in a variety of organisms, both procaryotic and eucaryotic. More recently this enzyme was found in bovine liver mitochondria. The mitochondrial purine nucleoside phosphorylase was purified 63 fold and has a molecular weight of 48–60 kD. From Lineweaver-Burk plots apparent K_m's of 23M for inosine, 42 M for deoxyinosine, 40 M for phosphate, 2 M for hypoxanthine, and 163 M for ribose-1-phosphate were calculated. Both 8-aminoguanosine (K_i=0.5 M) and araG (K_i=381 M) are inhibitors of the enzyme. The protein's isoelectric point (pI) was calculated at a pH of 4.2. Preliminary immunological work showed no cross-reactivity between epitopes on the mitochondrial protein and those on PNPase from human erythrocytes. The apparent K_m's calculated for the mitochondrial enzyme are,with the exception of that using hypoxanthine, within the range commonly associated with K_m's from the cytosolic species. The mitochondrial enzyme's molecular weight and pI are less than normally described. The enzyme's isolation from mitochondria, together with several unique characteristics, suggest that it is a separate protein from that found in the cytosol.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

P2Y6 receptor‐Gα12/13 signalling in cardiomyocytes triggers pressure overload‐induced cardiac fibrosis

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)‐β. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Gα_12/13) in cardiomyocytes suppressed pressure overload‐induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF‐β, connective tissue growth factor, and periostin) and Ang‐converting enzyme (ACE) was suppressed by the functional inhibition of Gα_12/13. The expression of these fibrogenic genes through Gα_12/13 by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G‐protein‐coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Gα_12/13 in cardiomyocytes by the extracellular nucleotides‐stimulated P2Y₆ receptor triggers fibrosis in pressure overload‐induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF‐β.     

Local stimulation of the adenosine A2B receptors induces an increased release of IL-6 in mouse striatum: an in vivo microdialysis study

Both adenosine and interleukin-6 (IL-6) have been implicated in the pathophysiology of, e.g., epileptic seizures, traumatic brain injury, and affective disorders. Stimulation of adenosine A_2B receptors on astrocytes in vitro leads to the increased synthesis and secretion of IL-6. We investigated whether or not activation of adenosine receptors evokes an increase of IL-6 release also in vivo . 5'- N -ethylcarboxamidoadenosine, a non-specific adenosine-agonist or vehicle was administered into the striatum of freely moving mice by reverse microdialysis. A statistical significant increase of the IL-6 concentration in the perfusate was detected already 60 min after 5'- N -ethylcarboxamidoadenosine administration. IL-6 increased progressively and reached a maximum after 240 min. This effect appears to be mediated through adenosine A_2B receptors since it was counteracted by the specific A_2B receptor antagonist MRS1706 but not by the specific A₁ receptor antagonist DPCPX. We conclude that adenosine via activation of A_2B receptors evokes IL-6 release also in vivo .     

Kinetics of luminol sonochemiluminescence quenched by purines

A homogeneous chemiluminescence (CL) reaction was initiated by ultrasound irradiation. Luminol sonochemiluminescence (SCL) reaction kinetics were determined under pseudo‐first‐order conditions, and the reaction followed the model for simple rise–fall kinetics. In addition, SCL quenching reactions induced by purines were also investigated in which the interactions between luminol and purines were analysed using the Stern–Volmer (S‐V) mechanism. The results implied that the high rate constant of luminol CL quenched by purines may be attributed to ground state interactions originating from hydrogen bonding. Copyright © 2012 John Wiley & Sons, Ltd.     

Antioxidant Status and Purine Bases in Carotid Artery Plaque

Free radical excess and oxidative stress are implicated in the formation and progression of atherosclerotic plaque through actions on susceptible vascular cells, such as by activating xanthine oxidase. Purine bases and other antioxidant compounds could play important protective roles in atherogenesis, as could nonenzymatic low molecular weight thiol defenses, not previously evaluated in carotid artery plaque. Therefore, we measured purine catabolites (hypoxanthine, xanthine, uric acid, allantoin) and antioxidant compounds (total sulphydryl groups, homocysteine, cysteine, and glutathione) in advanced carotid artery plaque and found a high ratio of allantoin to uric acid, suggesting a ongoing local oxidative stress.     

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Cell signal transduction: hormones,neurotransmitters and therapeutic drugs relate to purine nucleotide structure

Purine nucleotides transduce cell membrane receptor responses and modulate ion channel activity. This is accomplished through conformational change in the structure of nucleotides and cell membrane associated proteins. The aim of this study is to enhance our understanding of nucleotide dependence in regard to signal transduction events, drug action and pharmacological promiscuity. Nucleotides and ligand structures regulating Gα protein subunits, voltage- and ligand-gated ion channels are investigated for molecular similarity using a computational program. Results differentiate agonist and antagonist structures, identify molecular similarity within nucleotide and ligand structures and demonstrate the potential of ligands to regulate nucleotide conformational change. Relative molecular similarity within nucleotides and the ligands of the major receptor classes provides insight into mechanisms of receptor and ion channel regulation. The nucleotide template model has some merit as an initial screening tool in the study and comparison of drug and hormone structures.     

New Acyclic C-Nucleosides of the Imidazole

Abstract

Acid catalyzed isomerization of 1-aryl-(1,2-dideoxy-D-glycero-β-L-gluco-heptofuranose) [1,2-d]-2-imidazolines (4) yields 1-aryl-4-(D-galacto-pentitol-1-yl)imidazoles (8) which can be also obtained by reductive desulphuration of 1-aryl-2-benzylthio-4-(D-galacto-pentitol-1-yl)imidazoles (6). Compounds (4) were obtained by desulphuration with Raney nickel from 1-aryl-(1,2-dideoxy-D-glycero-β-L-gluco-heptofuranose) [1,2-d]-imidazolidine-2-thiones (1) or 1-aryl-2-benzylthio-(1,2-dideoxy-D-glycero-β-L-gluco-heptofuranose) [1,2-d]-2-imidazolines (2).     

Studies of the purine analog associated modulation of human erythrocyte acid phosphatase activity

Summary The activity of the human erythrocyte acid phosphatase is modulated by a series of structural analogs of purine. The unsubstituted purine base does not affect the enzyme activity. Addition of a substituent at the number six position usually generates an analog which activates the enzyme while similar substitutions at the two position usually generate an inhibitor. Pyrimidines are generally ineffective as modulators while several modifications of the imidazole ring of the purine analogs do not abolish the modulator activity of the purine analog. The level of response to all active analogs is isozyme specific. Differences in apparent relative affinities among the modulators are noted. The modulators with a positive effect on enzyme activity, are effective in the presence of methanol which is more effective than H₂0 as a phosphate acceptor. These analogs act by enhancing the rate of transfer of phosphate to H₂O, while decreasing the rate of transfer to methanol. The results suggest that the purine analogs may act by altering the rate of hydrolysis of the phosphoenzyme intermediate by H₂0 or may change the rate-limiting step in the catalytic mechanism.     

Fast DNA Interstrand Cross-Linking Reaction by 6-Vinylpurine

Oligonucleotides that incorporate a reactive moiety to form an interstrand cross-link have been widely studied for their potential toward inhibiting gene expression or as basic tools for chemical biology studies. The 6-vinylpurine (2) newly designed in the current study serves well as a new purine-base moiety for increasing cross-link reactivity to target cytosine. Thus, oligonucleotides containing 6-vinylpurine exhibit a more selective and much smoother DNA cross-linking ability to cytosine than the oligonucleotide analogs derived from 2-amino-6-vinylpurine (1) previously explored.     

The synthesis of <Emphasis Type="Italic">N</Emphasis><Superscript>2</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>6</Superscript>-substituted diaminopurine ribosides

A series of new 2,6-substituted diaminopurine riboside derivatives were synthesized by activation of protected xantosine with sulfonyl chlorides followed by treatment with various amines. The relationship between the reactivity of intermediates and the nature of the activating agents was studied.