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PubMed2000

Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation.

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Acetylglucosamine↗

PubMed1957

Effects of various immune rabbit serums on the cells of several transplanted mouse lymphomas in vitro and in vivo; with special reference to inhibition by an antibody that reacts with neoplastic and non-neoplastic lymphocytic cells of mice.

Immune serums prepared in rabbits with antigens made from normal mouse organs and tissues that were presumably devoid of large numbers of lymphocytic cells (notably kidney, liver, brain, whole embryos, and erythrocytes) proved lethal for the cells of several transplanted mouse lymphomas in vitro in the presence of complement; but these immune serums, when given intraperitoneally in large amounts to susceptible mice that had been implanted subcutaneously with lymphoma cells of one or another of several types, failed entirely to inhibit growth of the lymphoma cells in vivo. In contrast, immune serums made with cells procured from transplanted mouse lymphomas as antigens, and those made with cells from normal mouse thymus or lymph nodes, acted even more powerfully upon the several types of lymphoma cells in vitro than did the immune serums prepared with normal mouse organs, and when given intraperitoneally to implanted mice they brought about death of the lymphoma cells in vivo, the effect being to a considerable extent specific and referable to an antibody that reacts with neoplastic and non-neoplastic lymphocytic cells of mice, as absorption experiments disclosed. In comparative tests, furthermore, the anti-lymphoma serums acted more powerfully upon the lymphoma cells in vivo than did such chemotherapeutic agents as amethopterin, azaguanine, ethionine, azaserine, and 6-mercaptopurine, given singly or in various combinations in maximal tolerated amounts, though their effects were not so powerful as those exerted by normal guinea pig serum on lymphoma cells of two types that are susceptible to its action in vivo. The significance of the findings was briefly discussed.

Animals↗

PubMed1958

Effects of arsenic-azoproteins on mouse lymphoma cells in vivo; with observations on the effects of other anti-lymphoma agents, and on the susceptibility to these effects of lymphoma cells of various types.

Conjugates made by coupling diazotized arsanilic acid with one or another of a variety of proteins regularly brought about the complete regression of established 6C3HED lymphomas in living mice without perceptibly harming the latter, while untreated control animals regularly died with lymphomatosis. Histologic studies made plain that the lymphoma cells promptly die in mice treated with the arsenic-azoproteins, while those in untreated control animals continue to proliferate. Various inorganic and organic arsenicals (including arsanilic acid and 4-arsonophenyldiazotate) were essentially devoid of effect on the lymphoma cells in vivo, and this proved true as well of the proteins employed (serum albumins and globulins procured from several species, casein, and ovalbumin). Mixtures of arsanilic acid and the several proteins, various sulfur-azoproteins, and a number of other substances-viz., amethopterin, chlorambucil, 6-mercaptopurine, 8-azaguanine, azaserine, 6-azauracil, 5-fluorouracil, thioTEPA, and DON, each given in maximal tolerated amounts-also failed to influence notably the course of established 6C3HED lymphomas in vivo. Although readily overcoming Lymphoma E9514 cells growing in the subcutaneous tissues of susceptible mice, the arsenic-azoproteins had little or no effect once these cells had reached the livers and spleens of susceptible hosts. Furthermore the arsenic-azoproteins had little or no effect in vivo on the cells of Lymphoma AKRL1, L1210, and L4946. The findings were considered in relationship to the respective susceptibilities of several types of lymphoma cells to other anti-lymphoma agents-notably guinea pig serum, immune serums prepared in rabbits with mouse lymphoma cells as antigens, and a variety of chemical compounds. Taken together, the observations provide proof that lymphoma cells of various types, although resembling one another quite closely in growth characteristics following transplantation in susceptible hosts, and in morphology as disclosed by ordinary microscopy, differ notably in susceptibility to the effects of the several anti-lymphoma agents.

Animals↗

PubMed1986

Protection of mice against tetanus toxin by combination of two human monoclonal antibodies recognizing distinct epitopes on the toxin molecule.

Human B-lymphocytes were fused with the human lymphoblastoid B-cell line WI-L2-729 HF2. Hybridoma frequencies were in the range of 10(-5) when the mononuclear cells were (a) prestimulated with pokeweed mitogen (PWM), (b) fused with polyethyleneglycol (PEG), and (c) selected in a hypoxanthine-azaserine (HAza) containing medium. To generate monoclonal antibodies (MAb) specific for tetanus toxin (TToxin) human spleen cells were precultured with PWM plus tetanus toxoid (TToxoid) in two separate fusions. Two hybridomas were selected based on high binding activity using an enzyme-linked immunosorbent assay (ELISA) for TToxoid. Both hybridomas, cloned twice and designated anti-TT1 and anti-TT2, exhibited a near tetraploid karyotype and showed stable production of antibody (0.15 micrograms/ml) over several months. Using ELISA for fragments of TToxin and the immunoblotting technique, the two IgG1 monoclonal antibodies were found to bind to the heavy chain portion of the B-fragment (anti-TT1) and on the C-fragment (anti-TT2) of the toxin. When tested in an ELISA with TToxin the combination of anti-TT1 and anti-TT2 showed higher binding activity than either reagent alone. In an in vivo neutralization assay mice were completely protected against TToxin by the combination of the two antibodies while either antibody alone resulted only in a delay of death of the mice. These findings demonstrate that a cocktail of appropriate human monoclonal antibodies can be far superior to a single reagent when used in a therapeutic setting.

Animals↗

PubMed1989

Pancreatic acinar cell tumors in rats induced by 3,2'-dimethyl-4-aminobiphenyl.

Pancreatic acinar cell lesions, including foci, nodules, adenomas and in situ carcinomas, were found in male F344 rats given s.c. injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) at doses ranging from 50 to 167 mg/kg body weight in two 60-week experiments. The pancreatic lesions induced by DMAB were essentially the same as those reported in rats treated with 4-hydroxyaminoquinoline-1-oxide and azaserine. DMAB at high doses induced pancreas acinar cell as well as fat necrosis, and tumorigenicity for the pancreas was most effective at high necrogenic levels of carcinogen exposure. Mean labeling indices in basophilic foci, acidophilic foci, nodules and adenomas assessed by bromodeoxyuridine incorporation were elevated to approximately 0.69, approximately 2.17, approximately 1.43 and approximately 0.96% respectively, in contrast to the value of 0.39% observed for the surrounding normal acinar cells. The increase was significant in the acidophilic foci and nodules and the fact that these lesions had very high labeling indices suggests that they may have potential for progression to tumors.

Adenoma↗

PubMed1990

Role of cholecystokinin in the development of BOP-induced pancreatic lesions in hamsters.

Cholecystokinin (CCK) has been shown to promote pancreatic growth and azaserine-induced pancreatic carcinogenesis in rats. The present study was carried out to determine effects of CCK on pancreatic growth and carcinogenesis in the N-nitrosobis(2-oxopropyl)amine (BOP) hamster model. One hundred male Syrian golden hamsters were injected s.c. once weekly with 20 mg BOP/kg body wt at 6, 7 and 8 weeks of age, and divided into four groups of 25 animals each, which received one of the following treatments (once daily, 3 days/week for 16 weeks): gelatin; CR-1409, a potent CCK-receptor antagonist; CCK-8, 2.5 micrograms/kg body wt; or CCK-8 in combination with CR-1409 (30 min before CCK treatment). The animals were killed after 19 weeks. The growth of the pancreas but not the incidence of pancreatic (pre)neoplastic lesions was enhanced by CCK-8. CR-1409 did not influence the effect of CCK on pancreatic growth.

Animals↗

PubMed1991

Activation of c-K-ras is frequent in pancreatic carcinomas of Syrian hamsters, but is absent in pancreatic tumors of rats.

We have investigated the presence of mutations in ras genes at codons 12, 13 and 61 in chemically induced pancreatic tumors of rats and Syrian hamsters. Mutations were detected by means of allele-specific oligonucleotide hybridization to ras sequences amplified in vitro by the polymerase chain reaction. No mutations were observed in the c-K-ras gene or the c-H-ras gene of nine azaserine-induced adenomas and 15 carcinomas of the rat pancreas. This indicates that activated ras genes are not commonly involved in rat pancreatic cancer evolving from acinar cells. However, in 19 out of 20 ductular adenocarcinomas of hamster pancreas (95%), either codon 12 or 13 of the c-K-ras gene was mutated. This indicates that the activation of c-K-ras is a frequent event in the multistep process of pancreatic carcinogenesis induced by the alkylating carcinogen N-nitrosobis(2-oxopropyl)amine (BOP). The mutations of both codons were G----A transitions of the second base which is consistent with the type of mutation to be expected from DNA alkylation. Activation of the c-K-ras gene, therefore, may not only be a frequent but also an early event in hamster pancreas carcinogenesis. The frequent activation of the c-K-ras gene in both human and hamster pancreatic cancer emphasizes the relevance of BOP-induced pancreatic adenocarcinomas in Syrian hamsters as an experimental model system for studying human pancreatic cancer.

Adenocarcinoma↗

PubMed1973

Diaminopurine-resistant mutants of cultured, diploid human fibroblasts.

Clones of cells resistant to 2,6-diaminopurine were detected in skin fibroblast cultures derived from 13 of 21 normal humans of both sexes from 17 unrelated families. Almost all of the cultures that yielded mutants were chosen for further study from among a total of 83 surveyed because they displayed a slight resistance to low concentrations of diaminopurine. The incidences of mutant colonies ranged between about 10(-5) and 10(-4) per cell surviving prior mutagenic treatment with MNNG. The incidences of spontaneous mutants were about 10(-7) to 10(-5) in three unrelated cultures. Most independent mutants had distinctly reduced activity of adenine phosphoribosyltransferase but some had apparently normal amounts of activity. Two mutants from unrelated boys had little or no detectable enzyme activity and were unable to effectively use exogenous adenine for growth when purine biosynthesis was blocked with azaserine. Most mutants could utilize exogenous adenine, just as most azaguanine-resistant fibroblast mutants can utilize exogenous hypoxanthine, even when their hypoxanthine-guanine phosphoribosyltransferase activity is reduced. Diverse genetic changes conferred diaminopurine resistance but their specific natures are still undefined. Gross numerical or structural chromosome abnormalities were not observed in the mutants examined so far. Since at least one gene responsible for adenine phosphoribosyltransferase activity is on autosome No. 16 our results suggest that at least some of the cultures yielding mutants were heterozygous and that alleles conferring diaminopurine resistance may be frequent enough to comprise a polymorphism.

Adenine↗

PubMed1987

Induction of mouse oocyte maturation in vivo by perturbants of purine metabolism.

Previous studies have suggested a critical role for purines in the maintenance of mouse oocytes in meiotic arrest. If so, then disrupting specific purine metabolic pathways in vivo might induce the resumption of oocyte maturation. To test this hypothesis, immature mice were primed with pregnant mare's serum gonadotropin and 24 h later received, by i.p. injection, one of several drugs that inhibit specific enzymes in the purine metabolic pathways. Cumulus cell-enclosed oocytes were isolated from the ovaries at varying times after drug treatment and assessed for germinal vesicle breakdown (GVBD). The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid (MA) and bredinin (Br), each induced GVBD in a dose-dependent fashion, Br being the more effective agent. When the kinetics of oocyte maturation were examined, 71% of the oocytes from MA-treated mice had undergone GVBD 21-22 h after drug administration. Moreover, 100% GVBD was observed in oocytes from Br-treated mice after 6 h. The action of these drugs appeared to be a direct one and not mediated through stimulation of pituitary gonadotropin release or atresia. Azaserine, an inhibitor of de novo purine synthesis, also induced GVBD in a dose-dependent manner. However, sodium hadacidin and dl-alanosine, inhibitors of adenylosuccinate synthetase, failed to elicit a maturational response in oocytes in vivo. These data support an essential role for guanyl and/or xanthyl derivatives in the maintenance of meiotic arrest in vivo.

Animals↗

PubMed1997

Hypoxanthine regulation of oocyte maturation in the mouse: insights using hypoxanthine phosphoribosyltransferase-deficient animals.

In this study the effects of hypoxanthine (HX) on meiotic maturation were compared using oocytes from mice possessing a hypoxanthine phosphoribosyltransferase null mutation (HPRT-) and from the corresponding HPRT-competent background strain (HPRT+). Oocyte-cumulus cell complexes and cumulus cell-enclosed oocytes (oocytes cultured while enclosed by cumulus cells) from HPRT+, but not HPRT-, mice took up HX and contained significant levels of HPRT activity. In addition, FSH increased, and HX suppressed, the de novo synthesis of purines in HPRT+ complexes, whereas de novo synthesis was elevated in HPRT complexes and was unaffected by FSH or HX. After 3 h of HX treatment, lower frequencies of germinal vesicle breakdown (GVB) were observed in cumulus cell-enclosed than in denuded HPRT+ oocytes; however, identical frequencies of maturation were observed in denuded and cumulus cell-enclosed HPRT oocytes. This demonstrates a direct inhibitory action of HX on the oocyte that does not depend on salvage, plus an additional action of the cumulus cells that requires HPRT activity. Nevertheless, cumulus cells from HPRT- mice are capable of exerting an additional inhibitory action of dibutyryl cAMP (dbcAMP) on the oocyte. A kinetics analysis of FSH action on HX-arrested cumulus cell-enclosed HPRT+ and HPRT- oocytes revealed, first, that the inhibitory effect of the cumulus cells is transient and, second, that HPRT activity is not required for FSH induction of GVB in HX-arrested oocytes. When dbcAMP- or HX-arrested oocytes were treated with FSH, GVB was blocked to the same extent in HPRT- oocytes with the purine de novo synthesis inhibitor, azaserine, but this drug was less effective in HX-treated HPRT+ oocytes. These results confirm the importance of the de novo pathway in hormone-induced maturation and also support a role for purine salvage as an alternative source of nucleotide in this process.

Animals↗

PubMed2004

Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells.

Because adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP), we inquired whether HBP activation affects pancreatic beta-cell survival. Exposure of human islets to high glucose resulted in increased apoptosis of beta-cells upon serum deprivation that was reversed by azaserine. Also, glucosamine, a direct precursor of the downstream product of the HBP, increased human beta-cells apoptosis upon serum deprivation, which was reversed by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADGP), an inhibitor of protein O-glycosylation. These results were reproduced in RIN rat beta-cells. Glucosamine treatment resulted in inhibition of tyrosine-phosphorylation of the insulin receptor (IR), IRS-1, and IRS-2, which was associated with increased O-glycosylation. These changes caused impaired activation of the PI 3-kinase/Akt survival signaling that resulted in reduced GSK-3 and FOXO1a inactivation. BADGP reversed the glucosamine-induced reduction in insulin-stimulated phosphorylation of IR, IRS-1, IRS-2, Akt, GSK-3, and FOXO1a. Impaired FOXO1a inactivation sustained expression of the pro-apoptotic protein Bim, without affecting Bad, Bcl-XL, or Bcl-2 expression. These results indicate that hyperglycemia may increase susceptibility to apoptosis of human and rat beta-cell through activation of the HBP. Increased routing of glucose through this metabolic pathway results in impaired activation of the IR/IRSs/PI3-kinase/Akt survival pathway by induction of O-glycosylation of signaling molecules.

Apoptosis↗

PubMed1993

Inhibition of growth of a transplanted rat pancreatic acinar carcinoma with CCK-8.

Gastrointestinal hormones and neuropeptides are known to regulate growth of various normal gastrointestinal tissues and many cancers. Since cholecystokinin (CCK) is considered the most potent trophic factor for the exocrine pancreas, we studied its effect on growth of an acinar cell tumor, initially induced by azaserine and transplanted to the rat, in comparison with the normal pancreas. When tumors became palpable, rats were treated three times daily for 12 or 14 days with CCK-8 or NaCl 0.9% (controls) alone or in combination with the CCK receptor antagonist CR1409 (10 mg/kg) administered subcutaneously twice daily. Then tumors and pancreata were analyzed for their size, composition, and CCK receptors. Tumor volume, weight, and protein content, RNA, DNA, and enzymes decreased after CCK-8 treatment in a dose-dependent manner, the maximal effect being observed with 4-micrograms/kg treatment. This inhibitory effect was partially suppressed by CR1409, which by itself also reduced tumor growth, but to a lesser degree. CCK-8 exerted a stimulating effect on growth of the normal pancreas with low doses (1 and 2 micrograms/kg) and an inhibitory effect or no effect with a higher dose (4 micrograms/kg). CR1409 prevented this latter effect, but did not affect by itself the normal pancreas. These findings suggest that CCK-8 inhibits growth of an acinar cell tumor grafted to the rat; this effect is mediated by the occupation of specific CCK receptors present in high density on these cells. In contrast, CCK-8 exerts a biphasic effect on the normal pancreas as a function of its dose.

Animals↗

PubMed1999

Regulation of cholecystokinin-mediated amylase secretion by leptin in rat pancreatic acinar tumor cell line AR42J.

Expression of the long form of the leptin receptor, the isoform that is considered to have full signaling capability, has been reported in the central nervous system and several peripheral cell types. However, only a few cell lines have been shown to express the long form of the receptor. AR42J, a cell line derived from azaserine-treated rat pancreas, is a common model for pancreatic acinar cell secretion. In this study, the presence of leptin-receptor variants and leptin action was evaluated in this cell line. Messenger RNAs for both the long and a short form of the leptin receptor were detected by reverse transcription-polymerase chain reaction (RT-PCR) in AR42J cells, and authenticity of the receptor was confirmed by DNA sequencing. Competitive binding studies demonstrated that binding of radiolabeled leptin was specific and did not cross-react with cholecystokinin (CCK). Biologic effects of leptin on amylase release and intracellular calcium mobilization were further assessed in the presence and the absence of CCK, a known pancreatic secretagogue. Although leptin alone (< or =200 ng/ml) did not affect basal amylase release, it inhibited amylase release stimulated by 1 nM CCK by 48%. Leptin alone had no significant effect on calcium mobilization. However, pretreatment of leptin (10 and 100 ng/ml) enhanced calcium responses stimulated by CCK. These data demonstrate that the rat pancreatic tumor cell line AR42J expresses a functional form of the leptin receptor that modulates the action of CCK in calcium mobilization and amylase release.

Amylases↗

PubMed1984

Studies on sound-induced epilepsy in mice.

The cerebral concentrations of pyridoxal-5'-phosphate and divalent transition metal ions (Cu2+ and Zn2+) are appreciably higher in the seizure-susceptible strain of mouse (DBA/2J) than those in normal strains (CBA/Ca and Parkes ). By injecting metal ions intracranially and pyridoxal-5'-phosphate intraperitoneally, we could render the normal mouse prone to sound-induced epilepsy. The behaviour of the treated seizure-susceptible strain of mouse. The levels of glutamate and aspartate in its inferior colliculus were elevated and the concentration of gamma-aminobutyrate was lowered. Glutaminase inhibitors, 6-diazo-5-oxo-L-norleucine (DON) and 0-diazo-acetyl-L-serine (azaserine), and a transaminase inhibitor, 4-amino-3- isoxazolidone (L-cycloserine), when injected intraperitoneally, protected the seizure-susceptible mouse from undergoing convulsions, whereas pyridoxal-5'-phosphate and methionine sulphoximine, a glutamine synthetase inhibitor, exacerbated its epileptic condition. We propose a possible sequence of biochemical events associated with susceptibility to audiogenic seizures.

Acoustic Stimulation↗

PubMed1980

The reduction of nitrate to ammonium by a Clostridium sp. isolated from soil.

Cultures of Clostridium KDHS2 reduced 15NO3- to 15NH4+ with a concurrent increase in molar growth yield of 15.7% compared with fermentatively grown bacteria. The bacteria exhibited a Ks (NO3-) of 0.5 mM and reduced NO3- maximally at a rate of 0.1 mumol h(-1) mg dry wt)-1. A partially purified nitrate reductase was obtained which had a Km (NO3-) of 0.15 mM. The reduction of 13NO3- to 13NH4+ by resting bacteria was not inhibited by NH4+, glutamate, glutamine, methionine sulphoximine or azaserine. Glutamine synthetase affected neither the synthesis nor the activity of the NO3(-)-reducing enzymes. The results are consistent with the hypothesis that NO3- reduction to NH4+ in this Clostridium sp. is dissimilative. SO32-, but not SO42-, inhibited the reaction, apparently at the level of NO2- reduction.

Clostridium↗

PubMed1984

Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans.

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.

Amidohydrolases↗

PubMed1992

In situ nuclear magnetic resonance of N pulse labels monitors different routes for nitrogen assimilation.

Nuclear magnetic resonance offers the possibility of noninvasive in situ observation of (15)N pulse labeling in the presence of light. In vivo, exclusively the delta-nitrogen of Gln is labeled in the cyanobacterium Microcystis firma when glutamate synthase is inhibited by azaserine. In contrast, the green alga Chlorella fusca is additionally capable of incorporating nitrogen into Glu, thus providing evidence for an anabolic function of glutamate dehydrogenase in this organism.

Journal Article↗

PubMed1993

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica.

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.

Journal Article↗