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PubMed2000

Yeast GMP kinase mutants constitutively express AMP biosynthesis genes by phenocopying a hypoxanthine-guanine phosphoribosyltransferase defect.

We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.

Adenosine Monophosphate↗

PubMed1995

An investigation assessing the fraction of human immunodeficiency virus infection attributable to ethnic group variations in risk.

Considerable speculation and a recent publication has suggested that much of the human immunodeficiency virus (HIV-1) infection in the subset of the UK population represented by women having babies in Metropolitan London is substantially imported from Africa. Other data are supportive of this hypothesis. Ades et al. (1992) concluded that the fraction of HIV seroprevalence, identified in a large London-based study using neonatal blood from the dried blood spots on Guthrie cards, attributable to mothers born in Africa was 92.4% with 95% CI: 82-100%. This is an important observation which requires confirmation. This paper reports on an attempt to do this with closely similar methodology using the Unlinked Anonymous Survey (UAS) of HIV-1 seroprevalence in pregnant women attending antenatal centres, together with data from the Office of Population Censuses and Surveys (OPCS) on the country of origins of mothers delivering in 1991 at the 15 London centres included in the UAS. As in Ades' analysis there appeared to be a strong association between centre prevalences and the proportions of women of African origin delivering at those centres. This was consistent with a high fraction of seroprevalence attributable to such women, but it was also clear that the assumptions required for the estimation of the attributable fractions were not fully met. Analyses modified to justify the assumptions produced attributable fraction estimates ranging from 44% to 96% with lower 95% confidence limits 20% or less.(ABSTRACT TRUNCATED AT 250 WORDS)

Africa↗

PubMed1996

Neutralizing and infection-enhancing antibody responses to human immunodeficiency virus type 1 in long-term nonprogressors.

Serum antibodies from human immunodeficiency virus type 1 (HIV-1)-infected long-term non-progressors (LTNPs) and non-LTNPs were evaluated for virus neutralization and infection enhancement in vitro. Sera from LTNPs had higher average titers of neutralizing antibodies to HIV-1 strains IIIB and MN and more frequently neutralized primary isolates from progressors (14.9% vs. 1.3%, P = .002). Replication-competent HIV-1 was isolated from peripheral blood mononuclear cells and lymph nodes of 3 LTNPs. All viruses from LTNPs had a non-syncytium-inducing phenotype, were resistant to neutralization by autologous serum obtained at the time of virus isolation, and showed little evidence of a heightened sensitivity to neutralization by heterologous sera. Complement-mediated, antibody-dependent enhancement (C'-ADE) of HIV-1IIIB and primary isolates was equally prevalent for sera from LTNPs and non-LTNPs. Results indicate that LTNPs produce vigorous serum antibody responses and that long-term nonprogression is not associated with homologous neutralization or the absence of C'-ADE.

Adult↗

PubMed1988

Overview of postmarketing experience with ofloxacin in Germany.

Clinical trials with ofloxacin have shown that adverse drug events (ADEs) occurred in between 2.4% (Phase II) and 3.1-7.3% (Phase IV) of patients treated and were mostly mild. As with any other drug the true spectrum of rare events can only be fully appreciated after marketing. Since the launch of ofloxacin in June 1985 about 3.5 million patients have been treated in Germany, calculated on the basis of a mean daily dose of 400 mg ofloxacin and a mean duration of treatment of seven days. During these 2.5 years 985 spontaneous national reports of ADEs have been obtained and include rare adverse events (e.g. hallucination, psychotic reaction and shock), not seen in clinical trials. The present status of results from postmarketing surveillance is shown and discussed. The favourable overall risk:benefit ratio of ofloxacin appears unchanged.

Clinical Trials as Topic↗

PubMed1990

Purine levels and metabolism in human follicular fluid.

Adenosine (ADO) in low micromolar levels and hypoxanthine (HYP) in millimolar levels have been shown to inhibit maturation of cumulus-enclosed oocytes. To determine the effect of ovarian stimulation with gonadotrophins on follicular purine metabolism, we measured ADO, HYP, inosine (INO), adenine (ADE) and cyclic AMP (cAMP) levels in follicular fluid (FF) from natural (n = 7) or human menopausal gonadotrophin/human chorionic gonadotrophin (HMG/HCG)-stimulated (n = 35) cycles. Purines were extracted immediately (natural cycles) or within 30 min of recovery (HMG/HCG cycles) and analysed by high pressure liquid chromatography (HPLC). The concentration of all ADE purines in FF was in the low micromolar range (1-20 microM); cAMP levels were markedly increased (greater than 100 microM) in FF of HMG/HCG-treated patients. While ADO levels were within the range effective for inhibition of oocyte maturation, those of HYP were not. No correlation was found between purine levels in FF and ovum maturation. Purine levels in FF of natural cycles were uniformly lower than those of stimulated cycles. Significant conversion of 5'-AMP into ADO, ADO into INO and INO into HYP occurred within 1 h when FF was incubated at 25 but not at 4 degrees C. These purine levels in human FF confirm our previous findings with bovine FF and suggest a possible role of ADO, but not of HYP, in the inhibition of oocyte maturation in the human.

Adenine↗

PubMed2000

The rate of cell growth is regulated by purine biosynthesis via ATP production and G(1) to S phase transition.

We recently showed that an increased supply of purine nucleotides increased the growth rate of cultured fibroblasts. To understand the mechanism of the growth rate regulation, CHO K1 (a wild type of Chinese hamster ovary fibroblast cell line) and CHO ade (-)A (a cell line deficient in amidophosphoribosyltransferase, a rate-limiting enzyme of the de novo pathway) were cultured under various conditions. Moreover, a defective de novo pathway in CHO ade (-)A cells was exogenously restored by 5-amino-4-imidazole-carboxamide riboside, a precursor of the de novo pathway. The following parameters were determined: the growth rate of CHO fibroblasts, the metabolic rate of the de novo pathway, the enzyme activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the content of intracellular nucleotides, and the duration of each cell-cycle phase. We concluded the following: (i) Purine de novo synthesis, rather than purine salvage synthesis or pyrimidine synthesis, limits the growth rate. (ii) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine is available for energy conservation. (iii) The GTP content depends on the intracellular ATP content. (iv) Biosynthesis of purine nucleotides increases the growth rate mainly through ATP production and promotion of the G(1)/S transition.

Adenosine Triphosphate↗

PubMed2006

Cytogenetic damage induced by acrylamide and glycidamide in mammalian cells: correlation with specific glycidamide-DNA adducts.

Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite playing a central role in AA genotoxicity. In this work we investigated DNA damage induced by AA and GA in mammalian cells, using V79 Chinese hamster cells. For this purpose, we evaluated two cytogenetic end points, chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), as well as the levels of specific GA-DNA adducts, namely, N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) using high-performance liquid chromatography coupled with tandem mass spectrometry. GA was more cytotoxic and clastogenic than AA. Both AA and GA induced CAs (breaks and gaps) and decreased the mitotic index. GA induced SCEs in a dose-responsive manner; with AA, SCEs were increased at only the highest dose tested (2mM). A linear dose-response relationship was observed between the GA concentration and the levels of N7-GA-Gua. This adduct was detected for concentrations as low as 1 microM GA. N3-GA-Ade was also detected, but only at very high GA concentrations (>or= 250 microM). There was a very strong correlation between the levels of N7-GA-Gua in the GA- and AA-treated cells and the extent of SCE induction. Such correlation was not apparent for CAs. These data suggest that the induction of SCEs by AA is associated with the metabolism of AA to GA and subsequent formation of depurinating DNA adducts; however, other mechanisms must be involved in the induction of CAs.

Acrylamide↗

PubMed2006

The dopamine D3 receptor plays an essential role in alcohol-seeking and relapse.

Our study aimed to identify new candidate genes, which might be involved in alcohol craving and relapse. To find changes in gene expression after long-term alcohol consumption, we studied gene expression profiles in the striatal dopamine system by using DNA microarrays of two different alcohol-preferring rat lines (HAD and P). Our data revealed an up-regulation of the dopamine D3 receptor (D3R) after 1 yr of voluntary alcohol consumption in the striatum of alcohol preferring rats that was confirmed by qRT-polymerase chain reaction. This finding was further supported by the finding of up-regulated striatal D3R mRNA in nonselected Wistar rats after long-term alcohol consumption when compared with age-matched control animals. We further examined the role of the D3R in mediating alcohol relapse behavior using the alcohol deprivation effect (ADE) model in long-term alcohol drinking Wistar rats and the model of cue-induced reinstatement of alcohol-seeking behavior using the selective D3R antagonist SB-277011-A (0, 1, 3, and 10 mg/kg) and the partial agonist BP 897 (0, 0.1, 1, and 3 mg/kg). Both treatments caused a dose-dependent reduction of relapse-like drinking in the ADE model as well as a decrease in cue-induced ethanol-seeking behavior. We conclude that long-term alcohol consumption leads to an up-regulation of the dopamine D3R that may contribute to alcohol-seeking and relapse. We therefore suggest that selective antagonists of this pharmacological target provide a specific treatment approach to reduce alcohol craving and relapse behavior.

Alcohol Drinking↗

PubMed1997

Two parallel routes of the complement-mediated antibody-dependent enhancement of HIV-1 infection.

OBJECTIVE: To study the mechanism of the complement-mediated antibody-dependent enhancement (C'-ADE) of HIV infection which may play a significant role in the progression of HIV-disease. METHODS: In vitro complement activating and complement-mediated HIV-infection enhancing abilities of three human anti-gp41 monoclonal antibodies (MAb) were tested. C'-ADE was estimated using HIV-1IIIB and CR2 (CD21)-carrying MT-4 target cells. Normal human serum (NHS), purified C1q, C1q-deficient (C1qD) and C2-deficient (C2D) human sera were applied as complement sources. RESULTS: All MAb mediated increased C1q binding to solid-phase gp41. All MAb had a marked dose-dependent and strictly complement-mediated HIV-infection enhancing effect. Mixtures of the MAb with purified C1q also significantly increased HIV-1 infection. C1qD serum had a markedly lower enhancing effect than NHS, which could be raised to normal level by addition of purified C1q. Pretreatment of the target cells with anti-CR2 antibodies only partially inhibited the enhancing effect of the MAb plus normal human serum. CONCLUSION: These novel findings indicate that besides the well-known facilitation of entry of HIV-1 by the interaction between virus-bound C3 fragments and CR2 present on the target cells, fixation of C1q to intact virions also results in an enhanced productive HIV-1 infection in the MT-4 cell cultures.

Antibodies, Monoclonal↗

PubMed1998

Inactivation of a common epitope responsible for the induction of antibody-dependent enhancement of HIV.

BACKGROUND: The primary antigenic domain responsible for complement-mediated antibody-dependent enhancement (C'-ADE) of HIV and simian immunodeficiency virus resides in the principal immunodominant sequence of the transmembrane protein. OBJECTIVE: To identify whether there are amino-acid residues common to the epitopes of the known enhancing human monoclonal antibodies (MAb), and to provide a structural model for this functional region present on the HIV envelope. Since our model predicts that this region is involved in the association of gp120 with gp41, this association was monitored for each mutant. DESIGN: The binding of enhancing human MAb to point and deletion mutations within the enhancing domain was analyzed by two methods. The first analyzed binding to mutants expressed in COS cells: the second quantified the binding of four enhancing human MAb to each mutant gp160 versus wild-type control by enzyme-linked immunosorbent assay (ELISA). METHODS: Site-directed mutagenesis was used to produce specific deletions and point mutants, which were expressed in COS cells. Binding of MAb 50-69 and V3-loop MAb 5F7 were visualized in the wild-type and each of the mutant constructs by immunohistochemistry. Quantitative evaluation of enhancing human MAb binding to each mutant versus wild-type was performed by ELISA. A model for the enhancing domain and its relationship to gp120 association with gp41 was provided by molecular dynamics and ligand docking methods. RESULTS: All available enhancing human MAb known to bind to the principal immunodominant region of gp41 were unable to bind to deletions involving the disulfide loop, which in our molecular model provided the primary association site between gp120 and gp41. Point mutations in the loop blocked this association, but had a quantitatively smaller effect on the binding of the enhancing human MAb. A conservative W596Y mutation completely blocked the binding of all human MAb, but had no effect on gp120-gp41 association. CONCLUSIONS: A variety of mutations within the primary C'-ADE domain inhibit binding of enhancing human MAb as well as blocking the association of gp120 and gp41. A conservative W596Y mutation blocks binding of all enhancing human MAb with retention of gp120-gp41 association. These data are important to the design of vaccines in which the primary enhancing epitope is disarmed to prevent the subsequent induction of an amnestic response that could lead to viral enhancement of infection. The retention of the gp120-gp41 association is postulated to yield an immunogen similar to natural infection for both subunit and genetic vaccines.

Amino Acid Sequence↗

PubMed2000

Factors associated with clinical and virological failure in patients receiving a triple therapy including a protease inhibitor.

OBJECTIVE: To determine the predictors of virological and clinical failure in patients receiving a protease inhibitor as part of triple therapy. METHODS: From the French Hospital Database on HIV, 1402 protease inhibitor-naive patients starting a highly active antiretroviral therapy regimen with ritonavir, saquinavir-hard gel capsule (hgc) or indinavir between July 1996 and March 1997, and with measured HIV RNA at baseline and at 12 months, were studied for progression to a new AIDS-defining event (ADE) or death. Virological failure was defined as plasma HIV RNA > 1000 copies/ml at 12 months. Multivariate analyses were performed using Cox models for clinical outcomes and logistic regression for virological outcomes. RESULTS: During a median follow-up of 14.1 months, 94 (6.7%) patients experienced an ADE or died. At 12 months, 700 patients (49.9%) had virological failure. In the multivariate analysis, baseline CD4 cell count and viral load were found to be independent predictors of both virological and clinical failure. Neither the type of the first protease inhibitor taken nor previous antiretroviral therapy experience was associated with risk of clinical progression. For virological failure, the use of saquinavir-hgc was associated with a significant 1.96-fold increase in risk compared with indinavir; pre-treated patients were at higher risk than antiretroviral therapy-naive patients. CONCLUSION: In this study with large samples of patients, the use of saquinavir-hgc was associated with higher risk of virological failure at 12 months than were ritonavir and indinavir; no differences between protease inhibitors were found for clinical progression. As biases cannot be excluded, a longer duration of follow-up will be necessary to answer the question of whether the results are really discrepant or simply reflect the delay between virological failure and clinical manifestations.

Adult↗

PubMed2004

Immunologic and clinical responses to highly active antiretroviral therapy over 50 years of age. Results from the French Hospital Database on HIV.

OBJECTIVE: To study immunologic and clinical responses to HAART in patients over 50 years old. DESIGN AND METHODS: A prospective cohort study which included 68 hospitals in France. A total of 3015 antiretroviral-naive patients, 401 of whom were aged 50 years or over, were enrolled following initiation of HAART. The influence of age on the mean CD4 cell count increase on HAART was studied by using a two-slope mixed model. Progression, defined by the occurrence of a new AIDS-defining event (ADE) or death, was studied by Cox multivariate analyses. RESULTS: Among patients with baseline HIV RNA above 5 log copies/ml, CD4 mean increase during the first 6 months on HAART was +42.9 x 10(6) cells/l per month in patients under 50 years and +36.9 x 10(6) cells/l per month in patients over 50 years (P < 0.0001); subsequently, the respective monthly changes were +17.9 and +15.6 x 10(6) cells/l per month (P < 0.0001). Similar trends were observed in patients with baseline HIV RNA below 5 log copies/ml, and also after stratification for the baseline CD4 cell count. After a median follow-up of 31.5 months, 263 patients had a new ADE and 44 patients died. After adjustment for baseline characteristics, older patients had a significantly higher risk of clinical progression (hazard ratio (HR) = 1.52 [95% confidence interval (CI), 1.15-2.00]) and were more likely to achieve a viral load below 500 copies/ml [HR = 1.23, (95% CI, 1.11-1.38)]. CONCLUSION: Patients over 50 years of age have an immunologic response to HAART. However, their CD4 cell reconstitution is significantly slower than in younger patients, despite a better virologic response. This impaired immunologic response may explain their higher risk of clinical progression.

Aged↗

PubMed2000

Prevalence of dry eye subtypes in clinical optometry practice.

BACKGROUND: Dry eye conditions are now recognized as having multiple causes. A subtype-based dry eye diagnostic protocol was developed to determine the prevalence of dry eye and dry eye subtypes, and the effects of age and gender, in subjects presenting to clinical optometry practice. METHODS: Dry eye diagnostic criteria were: presence of one or more McMonnies dry eye survey primary symptoms, fluorescein tear break time < 10 s and rose bengal ocular surface staining. Dry eye subtype differential diagnosis was made predominantly on the basis of biomicroscopic signs. Subtype categories were: lipid anomaly dry eye (LADE), aqueous tear deficiency (ATD), primary mucin anomalies, allergic/toxic dry eye (ADE), primary epitheliopathies and lid surfacing/blinking anomalies (LSADE). RESULTS: Dry eye prevalence was 10.8% for n = 1584 subjects. Dry eye was significantly more prevalent in subjects 40 years or older (18.1%) compared with those < 40 years (7.3%) (p = 0.001). LADE was the most prevalent subtype (4.0%), followed by ADE at 3.1%, LSADE at 1.8%, and ATD at 1.7%. ATD was the only subtype with a significant gender prevalence difference, being more prevalent in women (p = 0.0023). The prevalence of LADE and ATD were significantly greater in those 40 years or older (p = 0.001 and p = 0.0023 respectively). CONCLUSIONS: The results of this study support a subtype-based approach to dry eye diagnosis and management in clinical practice.

Adolescent↗

PubMedDate not supplied

OSF healthcare's journey in patient safety.

This article describes OSF Healthcare's recent journey in patient safety. It discusses the involvement of its 6 hospitals in collaboration with each other and the Institute for Healthcare Improvement. OSF focused on a strategy for decreasing adverse drug events (ADEs). They worked on impacting 4 major areas: safety culture, high-risk medication, dispensing of medication, and medication reconciliation. By doing this, OSF decreased its rate of ADEs per 1000 units of medication administered from 3.84 to 1.39 over the course of 20 months.

Delivery of Health Care, Integrated↗

PubMed2003

Role of Fyn tyrosine kinase in ethanol consumption by mice.

BACKGROUND: Mice deficient for the intracellular protein Fyn tyrosine kinase (fynZ/fynZ mice) have been reported to show increased alcohol sensitivity and lack of tolerance to the effects of ethanol. To further study the involvement of Fyn in neurobehavioral effects of alcohol, we examined ethanol consumption and relapse drinking behavior in fynZ/fynZ mice. METHODS: FynZ/fynZ and wild-type mice were given a free choice between water and increasing concentrations of ethanol (2-16%). Once a stable baseline of 16% ethanol consumption was established, access to ethanol was withdrawn for 2 weeks and then reinstated, to measure the alcohol deprivation effect (ADE). Forced swim stress was performed thereafter on 2 consecutive days. In a final experiment we studied alcohol sensitivity by measuring ethanol-induced loss of righting reflex (LORR). RESULTS: The concentration of available ethanol had a significant effect on ethanol consumption and preference; however, there was no significant effect of genotype on these measures. Deprivation from ethanol led to a significant increase in ethanol consumption by all mice with no significant impact of genotype on ethanol consumption or water consumption during the ADE. Two consecutive days of forced swim stress led to a significant increase in ethanol consumption; again however, genotype had no effect on stress-associated ethanol consumption. Surprisingly, however, FynZ/fynZ mice showed no differences in alcohol sensitivity when compared to wild-type animals, in contrast to previously reported results ( Miyakawa et al., 1997). CONCLUSIONS: Deletion of the Fyn tyrosine kinase gene may be involved in ethanol sensitivity but this effect may depend on a gene-environment interaction. Fyn does not influence ethanol consumption, neither under basal conditions nor following a deprivation period or stress. This finding indicates that phosphorylation and activation of N-methyl-D-aspartate (NMDA) receptors through Fyn is not a critical mechanism in alcohol drinking or relapse behavior.

Alcohol Drinking↗

PubMed2003

Why are dengue virus serotypes so distantly related? Enhancement and limiting serotype similarity between dengue virus strains.

Dengue virus, the causative agent of dengue fever, has four major serotypes characterized by large genetic and immunological distances. We propose that the unusually large distances between the serotypes can be explained in the light of a process of antibody-dependent enhancement (ADE) leading to increased mortality. Antibody-dependent enhancement results from a new infection with a particular serotype in an individual with acquired immunity to a different serotype. Classical dengue fever causes negligible mortality, but ADE leads to the risk of developing the significantly more dangerous dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). A mathematical model is presented that describes the epidemiological dynamics of two serotypes of a pathogen where there is the possibility of co-infection and reinfection by a different serotype, along with increased mortality as a result of enhancement. We show that if there is no or slightly increased mortality after reinfection (enhancement), serotypes with a small immunological distance can stably coexist. This suggests that a cloud of serotypes with minor serological differences will constitute the viral population. By contrast, if enhancement is sufficiently great, a substantial immunological distance is necessary for two serotypes to stably coexist in the population. Therefore, high mortality owing to enhancement leads to an evolutionarily stable viral community comprising a set of distantly separated serotypes.

Antibody-Dependent Enhancement↗

PubMed2003

Cell fate decisions within the mouse organizer are governed by graded Nodal signals.

It is well known that cell fate decisions in the mouse organizer region during gastrulation ultimately govern gut formation and patterning, left-right axis determination, and development of the central nervous system. Previous studies suggest that signaling pathways activated by Nodal, bone morphogenetic protein (BMP), and Wnt ligands coordinately regulate patterning of the streak and the formation of midline organizing tissues, but the specific contributions of these molecules within discrete cell lineages are poorly defined. Here we removed Smad2 activity in the epiblast, using a conditional inactivation strategy. Abrogation of Smad2 does not compromise primitive streak (PS) formation or gastrulation movements, but rather results in a failure to correctly specify the anterior definitive endoderm (ADE) and prechordal plate (PCP) progenitors. To selectively lower Nodal activity in the posterior epiblast, we generated a novel allele lacking the proximal epiblast enhancer (PEE) governing Nodal expression in the PS. As for conditional inactivation of Smad2, germ-line deletion of the PEE selectively disrupts development of the anterior streak. In striking contrast, the node and its midline derivatives, the notochord and floor plate, develop normally in both categories of mutant embryos. Finally, we show that removal of one copy of Smad3 in the context of a Smad2-deficient epiblast results in a failure to specify all axial midline tissues. These findings conclusively demonstrate that graded Nodal/Smad2 signals govern allocation of the axial mesendoderm precursors that selectively give rise to the ADE and PCP mesoderm.

Alleles↗

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↗