Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

Purine and pyrimidine metabolism in the Trypanosomatidae

The pathways leading to purine and pyrimidine nucleotide production in members of the family Trypanosomatidae are discussed with special emphasis on data relating to pathogenic species published from 1974 to 1983 inclusive. Trypanosomes and leishmania in general lack a de novo purine biosynthetic pathway, but have a multiplicity of possible routes for purine salvage. In contrast, pyrimidine nucleotides can be produced by either de novo or salvage pathways. The properties of these pathways in trypanosomatids are compared and contrasted with those of their hosts.     

Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.     

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.     

Purine metabolism in Trypanosoma cruzi

Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rate-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine > hypoxanthine > guanine > xanthine.     

Purine metabolism in lymphocytes from patients with primary hypogammaglobulinaemia

The previous report of low levels of purine 5'-nucleotidase activity in peripheral blood mononuclear cells (lymphocytes and monocytes) from patients with non-familial adult onset `variable' primary hypogammaglobulinaemia has been confirmed and the observation extended to include patients with other types of primary immunodeficiency. Patients with sex-linked congenital hypogammaglobulinaemia have values for mononuclear cell 5'-nucleotidase activity which are in the normal range, whereas most cases of non-familial adult onset `variable' primary hypogammaglobulinaemia have clearly subnormal values. The three patients with isolated IgA deficiency who were tested also had subnormal values. Evidence that the measured enzyme activity is in fact 5'-nucleotidase and independent of interfering phosphatase activities is presented. No significant or consistent alterations in the activities of the following enzymes were detected in mononuclear cells or erythrocytes: adenosine deaminase, purine nucleoside (inosine) phosphorylase, hypoxanthine phosphoribosyltransferase, adenine phosphoribosyltransferase, phosphoribosylpyrophosphate (PRPP) synthetase. The erythrocyte PRPP content and the mononuclear cell PRPP amidotransferase activity were normal in the small number of patients in which they were measured. These findings are discussed in the light of the current interest in the inter-relationship between some disorders of purine metabolism and the immunological deficiency syndromes.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Purine nucleotide metabolism in resident and activated rat macrophages in vitro

The overall purine metabolism was studied in detail in resident peritoneal macrophages (M phi) and in thioglycolate elicited peritoneal M phi in vitro. The salvage of purine bases (adenine, hypoxanthine and guanine) was active in both M phi populations, whereas purine biosynthesis de novo was low. Purine nucleosides (inosine, guanosine and adenosine) were efficiently degraded to uric acid and only adenosine was directly salvaged into nucleotides. Purine salvage was markedly increased in elicited M phi as compared to resident M phi whereas purine degradation pathways were enhanced only slightly. These results clearly indicate that salvage of purine bases is the main source for purine nucleotide biosynthesis in M phi, but nucleotide catabolism is the predominant pathway.     

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.     

Purine metabolism by the avian malarial parasite Plasmodium lophurae

Extracts of normal duckling erythrocytes catabolized AMP to IMP, inosine and hypoxanthine; adenosine and adenine were not formed from AMP. When erythrocyte-free Plasmodium lophurae, prepared by antibody lysis, were incubated in the presence of [¹⁴C]hypoxanthine approximately 60% of the label was recovered as purine nucleotides and there was no evidence for extracellular alteration of added hypoxanthine. However, when adenosine was added to suspensions of antibody- or saponin-prepared parasites extensive conversion to inosine and hypoxanthine occurred. This conversion was found to be the result of parasite lysis with release of cytosolic purine salvage pathway enzymes; plasmodial surface membrane ecto-enzymes were not responsible for adenosine catabolism. It appears that in vivo the intracellular plasmodium utilizes the normal erythrocytic process of purine turnover to avail itself of hypoxanthine, the red cell's end-product, and at the same time the parasite avoids direct competition for adenosine essential to erythrocyte survival. Since the blood plasma of infected ducklings contained increased amounts of hypoxanthine it is possible that P. lophurae also utilizes this as a purine source.     

Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.     

Modulation of J774.1 macrophage L-arginine metabolism by intracellular Mycobacterium bovis BCG

Using a Mycobacterium bovis BCG mutant (AS1) lacking a Bacillus subtilis L-arginine transporter homolog, we demonstrate here that the interaction between intracellular mycobacteria and the macrophage with respect to L-arginine transport and metabolism is quite complex. Intracellular AS1 stimulates macrophage L-arginine transport and accumulates 2.5-fold more (3)H label derived from L-arginine than does the wild type. These studies suggest that the accumulation of (3)H label reflects the acquisition of metabolites of L-arginine produced by the macrophage.     

Purine Metabolic Enzymes in Lymphocytes

Coformycin, which is an inhibitor of adenosine deaminase, significantly inhibited in vitro blastogenic responses of human lymphocytes to both phytohaemagglutinin (PHA) and pokeweed mitogen (PWM), whereas blastogenic responses to bacterial lipopolysaccharide (LPS) were rather enhanced by the addition of coformycin. Blastogenic responses of lymphocytes to PHA and PWM were markedly suppressed by the addition of adenosine, which is a substrate of adenosine deaminase. Allopurinol, which is an inhibitor of xanthine oxidase, inhibited blastogenic responses of human lymphocytes to PHA, PWM, and bacterial LPS. Inosine (a substrate of purine nucleoside phosphorylase) and hypoxanthine (a substrate of xanthine oxidase) showed no or only a small effect on blastogenic responses of human lymphocytes. These results suggest that adenosine deaminase activity is associated with the T-cell response but not with the B-cell response and that the impaired T-cell response in adenosine deaminase deficiency is the result of intracellular retention of adenosine in T cells. The results also suggest that purine nucleoside phosphorylase or xanthine oxidase activity is associated with both T- and B-cell responses.     

Streptococcus bovis Meningitis and Hemorrhoids

We report a case of Streptococcus bovis (Streptococcus gallolyticus subsp. pasteurianus) meningitis, a rare cause of central nervous system (CNS) infection in an adult, and comment on the importance of investigation of the lower gastrointestinal tract to identify a portal of entry in cases of systemic Streptococcus bovis infection.     

Peroperative cell salvage

Red cell salvage (RCS) is now an established therapy for patients undergoing surgery with an associated risk of heavy bleeding. It should always be available for such patients. It is, however, only one part of what should be a multifaceted approach to blood conservation and it does not replace the need for good quality surgical haemostasis.