Salvage Metabolism Research Articles

Effect of pH on antibiotic resistance genes removal and bacterial nucleotides metabolism function in the wastewater by the combined ferrate and sulfite treatment

The variations of the antibiotic resistance genes removal and the bacterial nucleotide metabolism function in the wastewater by the combined ferrate and sulfite treatment at different pH levels were investigated. The total DNA as well as the genes sul2, sul3 and intI1 removal by the combined 10 mg/L ferrate and 28.5 mg/L sulfite treatment and the contributions of the sulfate radicals and the hydroxyl radicals produced by the reaction of ferrate with sulfite to the DNA removal decreased as the pH increase from 6.0 to 9.0. Bacterial nucleotide metabolism level changes affected the DNA removal, and the highest nucleotide degradation metabolism level and the lowest level of the nucleotide biosynthesis and salvage metabolism were found at pH 6.0. The reduction of the genes sul2, sul3 and intI1 host level contributed to the genes sul2, sul3 and intI1 removal. This study provides novel insights into the antibiotic resistance genes removal by the combined ferrate and sulfite treatment at different pH levels.

Possible Genetic Risks from Heat-Damaged DNA in Food.

The consumption of foods prepared at high temperatures has been associated with numerous health risks. To date, the chief identified source of risk has been small molecules produced in trace levels by cooking and reacting with healthy DNA upon consumption. Here, we considered whether the DNA in food itself also presents a hazard. We hypothesize that high-temperature cooking may cause significant damage to the DNA in food, and this damage might find its way into cellular DNA by metabolic salvage. We tested cooked and raw foods and found high levels of hydrolytic and oxidative damage to all four DNA bases upon cooking. Exposing cultured cells to damaged 2'-deoxynucleosides (particularly pyrimidines) resulted in elevated DNA damage and repair responses in the cells. Feeding a deaminated 2'-deoxynucleoside (2'-deoxyuridine), and DNA containing it, to mice resulted in substantial uptake into intestinal genomic DNA and promoted double-strand chromosomal breaks there. The results suggest the possibility of a previously unrecognized pathway whereby high-temperature cooking may contribute to genetic risks.

After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.

CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.

112P Clinicopathological characterization of MTAP-altered metastatic gastrointestinal tumors

Methyl-thio-adenosine phosphorylase (MTAP) is an essential enzyme for the purine salvage metabolism. In cancer, MTAP deficiency (MTAPdef) confers susceptibility to novel agents inhibiting the protein arginine methyl-transferase 5 (PRMT5) pathway. MTAPdef is a recurrent event in tumors with CDKN2A/B loss as these genes are closely located in chromosome 9p. In this study, we aim at investigating the main clinical characteristics of patients with gastrointestinal (GI) cancers harboring MTAP alterations.

Fusobacterium nucleatum promotes the development of acute liver failure by inhibiting the NAD+ salvage metabolic pathway

BackgroundAcute liver failure (ALF) patients are often accompanied by severe energy metabolism abnormalities and intestinal microecological imbalance. The intestinal mucosal barrier is severely damaged. Intestinal endotoxin can induce intestinal endotoxemia through the "Gut-Liver axis". More and more evidence shows that members of the gut microbiota, especially Fusobacterium nucleatum (F. nucleatum), are related to inflammatory bowel disease, but whether F. nucleatum is involved in the development of ALF and whether it affects the liver energy metabolism is unclear.MethodsThis study first detected the abundance of F. nucleatum and its effect on ALF disease, and explored whether F. nucleatum aggravated liver inflammation in vitro and in vivo.ResultsOur data showed that liver tissues of ALF patients contained different abundances of F. nucleatum, which were related to the degree of liver inflammation. In addition, we found that F. nucleatum infection affected the energy metabolism of the liver during the development of ALF, inhibited the synthesis pathway of nicotinamide adenine dinucleotide (NAD+)'s salvage metabolism, and promoted inflammatory damage in the liver. In terms of mechanism, F. nucleatum inhibited NAD+ and the NAD+-dependent SIRT1/AMPK signaling pathway, and promoted liver damage of ALF.ConclusionsFusobacterium nucleatum coordinates a molecular network including NAD+ and SIRT1 to control the progress of ALF. Detection and targeting of F. nucleatum and its related pathways may provide valuable insights for the treatment of ALF.

Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia reperfusion injury

Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways.

Differential susceptibility of human neural progenitors and neurons to ischaemic injury

BackgroundNeuroprotection for stroke has shown great promise but has had little translational success. Developing drugs for humans logically requires human tissue evaluation. Human embryonic stem cell (hESC)-derived neuronal cultures at different developmental stages were subject to oxygen glucose deprivation (OGD) to determine how developing maturity altered response to ischemic injury. MethodsH9 hESCs were induced by Noggin to generate neural progenitors (NPs) and highly arbourised structurally complex neurons. They were both subjected to OGD or OGD with reoxygenation (OGD-R) for 1−6 h.Outcome was assessed by measures of cell death, survival and morphology. ResultsNPs did not die after OGD but experienced progressive loss of metabolic activity. Highly arbourised neurons showed minimal cell death initially but 44 % and 78 % died after 4 and 6 h OGD. Metabolic dysfunction was greater in these more mature neurons (∼70 %) than in NPs and evident after 1 h OGD, before detection of neuronal death at 4 h. OGD-R salvaged metabolic activity but not cell death in mature neurons. In NPs there was little metabolic salvage and cell death was induced (50 % and 65 % at 4 and 6 h OGD-R, respectively). ConclusionsHighly arbourised neurons are more sensitive to ischaemic injury than NPs which did however develop marked vulnerability to prolonged injury with reoxygenation. These observations imply that therapeutic potential may be highly dependent of the developmental state of the neurons we aim to protect.

Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease.

The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction.

Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease.

ABSTRACTFamilial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). We found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. We conclude that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+, is a viable approach to preventing neurotoxicity associated with mitochondrial defects.

Abstract 4925: Targeted metabolic profiling of gliomas and glioma stem cells identifies methionine as a differentially regulated metabolite: Proof of principle supporting methionine-pet imaging

Abstract Purpose: At present, 18F-Fluorodeoxyglucose(FDG)-positron emission tomography (PET) is the most commonly used diagnostic imaging modality in clinical practice for predicting prognosis in malignant brain tumors. The diffusive nature of glioblastoma (GBM) often makes FDG-PET detection inaccurate and there is no diagnostic gold-standard for GBM. Thus, it is necessary to find new criteria independent of conventional FDG-PET diagnosis to delineate tumor versus normal tissues. Molecular imaging using 11C-Methionine-PET (Met-PET) is one of the most promising approaches to this challenge. Methods: We employed a targeted metabolomic approach to study the extracellular and intracellular levels of methionine in established (ATCC) and patient derived primary glioblastoma (GBM) cell lines, glioma stem cells (GSC) and normal human astrocytes (NHA) to gain a better understanding of methionine uptake. Methylthioadenosine phosphorylase (MTAP) is an enzyme that plays a vital role in polyamine metabolism and methionine salvage. Therefore, we determined the expression profile of MTAP in these cells using RT-qPCR. We then extracted the intracellular and extracellular metabolites of GBM, GSC and NHA to estimate their methionine levels using LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments. Thus, by using metabolic fingerprints of methionine, we quantified methionine accumulation / uptake in GBM, GSC and NHA. Results and Conclusion: PET provides real-time information of metabolism through the behavior of single molecules. Despite clinical trials advocating Met-PET imaging as a superior imaging modality compared to FDG-PET for brain tumors, to date Met-PET has not been widely used in clinical diagnostics. Through this metabolic profiling of gliomas, we have shown for the first time that GBM and GSC accumulate methionine when compared to NHA. Uptake studies through exogenous addition of methionine in GBM, GSC and NHA show a significant uptake of methionine by GBM and GSC when compared to NHA. The results of our study establish the scientific basis for Met-PET imaging in GBM and provide a rationale for incorporating Met-PET imaging in future brain tumor clinical trials to evaluate its prognostic value. Citation Format: Kamalakannan Palanichamy, Thirumoorthy Krishnan, Nikhil Sebastian, Rajbir Singh, Suman Kanji, Arnab Chakravarti. Targeted metabolic profiling of gliomas and glioma stem cells identifies methionine as a differentially regulated metabolite: Proof of principle supporting methionine-pet imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2014-4925

Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme

Homotrimeric mammalian purine nucleoside phosphorylase (PNP) plays a key role in the nucleoside and nucleotide metabolic salvage pathway. Each monomer in the active PNP trimer is composed of a central β-sheet flanked by several α-helices. We investigated the stability of calf PNP using analytical ultracentrifugation, differential scanning calorimetry, circular dichroism, and UV absorption spectroscopy. The results demonstrate that the activity decline (due to protein aging after isolation from cells) of wild type PNP and its two mutants with point mutations in the region of monomer–monomer interface, is accompanied by a decrease of the population of the trimeric enzyme and an increase of the population of its aggregated forms. The data do not indicate a significant population of either folded or unfolded PNP monomers. The enzyme with specific activity lower than the maximal shows a decrease of the helical structure, which can make it prone to aggregation. The presence of phosphate stabilizes the enzyme but leads to a more pronounced aggregation above the melting temperature. These results suggest that the biological role of packing of the PNP monomers into a trimeric structure is to provide the stability of the enzyme since the monomers are not stable in solution.

Stratification of Nucleoside Analog Chemotherapy Using 1-(2′-Deoxy-2′-18F-Fluoro-β-d-Arabinofuranosyl)Cytosine and 1-(2′-Deoxy-2′-18F-Fluoro-β-l-Arabinofuranosyl)-5-Methylcytosine PET

The ability to measure tumor determinants of response to nucleoside analog (NA) chemotherapy agents such as gemcitabine and related compounds could significantly affect the management of several types of cancer. Previously we showed that the accumulation in tumors of the new PET tracer 1-(2'-deoxy-2'-(18)F-fluoro-β-d-arabinofuranosyl)cytosine ((18)F-FAC) is predictive of responses to gemcitabine. (18)F-FAC retention in cells requires deoxycytidine kinase (dCK), a rate-limiting enzyme in the deoxyribonucleoside salvage metabolism and in gemcitabine conversion from an inactive prodrug to a cytotoxic compound. The objectives of the current study were to determine whether (18)F-FAC tumor uptake is also influenced by cytidine deaminase (CDA), a determinant of resistance to gemcitabine; to develop a new PET assay using (18)F-FAC and the related probe 1-(2'-deoxy-2'-(18)F-fluoro-β-l-arabinofuranosyl)-5-methylcytosine (l-(18)F-FMAC) to profile tumor lesions for both dCK and CDA enzymatic activities; and to determine whether this PET assay can identify the most effective NA chemotherapy against tumors with differential expression of dCK and CDA. Isogenic murine leukemic cell lines with defined dCK and CDA activities were generated by retroviral transduction. A cell viability assay was used to determine the sensitivity of the isogenic cell lines to the dCK-dependent NA prodrugs gemcitabine and clofarabine. In vitro enzymatic and cell-based tracer uptake assays and in vivo PET with (18)F-FAC and l-(18)F-FMAC were used to predict tumor responses to gemcitabine and clofarabine. dCK and CDA activities measured by kinase and tracer uptake assays correlated with the sensitivity of isogenic cell lines to gemcitabine and clofarabine. Coexpression of CDA decreased the sensitivity of dCK-positive cells to gemcitabine treatment in vitro by 15-fold but did not affect responses to clofarabine. Coexpression of CDA decreased (18)F-FAC but not l-(18)F-FMAC, phosphorylation, and uptake by dCK-positive cells. (18)F-FAC and l-(18)F-FMAC PET estimates of the enzymatic activities of dCK and CDA in tumor implants in mice were predictive of responses to gemcitabine and clofarabine treatment in vivo. These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for guiding the selection of NA prodrugs.

Cloning of Rabbit <italic>HPRT</italic> Gene Using the Recombineering System

Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

AMINOHYDROLASES ACTING ON ADENINE, ADENOSINE AND THEIR DERIVATIVES

Adenine and adenosine-acting aminohydrolases are important groups of enzymes responsible for the metabolic salvage of purine compounds. Several subclasses of these enzymes have been described and given current knowledge of the full genome sequences of many organisms, it is possible to identify genes encoding these enzymes and group them according to their primary structure. This article is a short overview of the enzymes classified as adenine and adenosine deaminase. It summarises knowledge of their occurrence, genetic basis and their catalytic and structural properties. These enzymes are constitutive components of purine metabolism and their impairment may cause serious medical disorders. In humans, adenosine deaminase deficiency is linked to severe combined immunodeficiency and as such the enzyme has been approved for the first gene therapy trial. The role of these enzymes in plants is unclear, since the activity was has not been detected in extracts and putative genes have not been yet cloned and analyzed. A literature search and amino acid identity comparison show that Ascomycetes contain only adenine deaminase, but not adenosine deaminase, despite the fact that corresponding genes are annotated in databases as the adenosine cleaving enzymes because they share the same conserved domain.

Residues 334 and 338 in Transmembrane Segment 8 of Human Equilibrative Nucleoside Transporter 1 Are Important Determinants of Inhibitor Sensitivity, Protein Folding, and Catalytic Turnover

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.

Differential gene expression of GDP‐L‐fucose‐synthesizing enzymes, GDP‐fucose transporter and fucosyltransferase VII

L-fucose is a fundamental monosaccharide component of many mammalian glycoproteins and glycolipids. Fucosylation requires GDP-L-fucose as a donor of fucose and a specific fucosyltransferase (Fuc-T) to catalyze the transfer of L-fucose to various lactosamine acceptor molecules. The biosynthesis of GDP-L-fucose consists of two pathways. The constitutively active de novo pathway involves conversion of cellular GDP-D-mannose to GDP-L-fucose by GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (FX). In the alternative biosynthetic pathway, in the salvage metabolism, L-fucokinase (Fuk) synthesizes L-fucose-1-phosphate from free fucose. L-fucose-1-phosphate is further catalyzed to GDP-L-fucose by GDP-L-fucose pyrophosphorylase (Fpgt). GDP-L-fucose, synthesized in the cytosol, is translocated to the Golgi for fucosylation by a specific GDP-fucose transporter (FUCT1). Glycans that contain alpha(1,3)-fucosylated modifications, e.g. sialyl Lewis X-type glycans, have an important role in inflammation and in tumorigenesis. We studied the mRNA expression levels of GDP-L-fucose-synthesizing enzymes, GDP-fucose transporter and fucosyltransferase VII by quantitative real-time PCR in mouse endothelial cells, macrophages and lymphoid tumor cells. Moreover, the expression of the same transcripts was detected in acute inflammation using rat kidney allograft as model system. Our results indicate the simultaneous upregulation of the GDP-L-fucose synthesizing enzymes of the de novo pathway, GDP-fucose transporter and fucosyltransferase VII in inflammation and in tumorigenesis.

Three-dimensional structure of human adenine phosphoribosyltransferase and its relation to DHA-urolithiasis.

In mammals, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) is present in all tissues and provides the only known mechanism for the metabolic salvage of adenine resulting from the polyamine biosynthesis pathway or from dietary sources. In humans, APRT deficiency results in serious kidney illness such as nephrolithiasis, interstitial nephritis, and chronic renal failure as a result of 2,8-dihydroxyadenine (DHA) precipitation in the renal interstitium. To address the molecular basis of DHA-urolithiasis, the recombinant human APRT was crystallized in complex with adenosine 5'-monophosphate (AMP). Refinement of X-ray diffraction data extended to 2.1 A resolution led to a final crystallographic R(factor) of 13.3% and an R(free) of 17.6%. This structure is composed of nine beta-strands and six alpha-helices, and the active site pocket opens slightly to accommodate the AMP product. The core of APRT is similar to that of other phosphoribosyltransferases (PRTases), although the adenine-binding domain is quite different. Structural comparisons between the human APRT and other "type I" PRTases of known structure revealed several important features of the biochemistry of PRTases. We propose that the residues located at positions corresponding to Leu159 and Ala131 in hAPRT are responsible for the base specificities of type I PRTases. The comparative analysis shown here also provides structural information for the mechanism by which mutations in the human APRT lead to DHA-urolithiasis.

Ganglioside/glycosphingolipid turnover: new concepts.

In this review focus is given to the metabolic turnover of gangliosides/glycosphingolipids. The metabolism and accompanying intracellular trafficking of gangliosides/glycosphingolipids is illustrated with particular attention to the following events: (a) the de novo biosynthesis in the endoplasmic reticulum and Golgi apparatus, followed by vesicular sorting to the plasma membrane; (b) the enzyme-assisted chemical modifications occurring at the plasma membrane level; (c) the internalization via endocytosis and recycling to the plasma membrane; (d) the direct glycosylations taking place after sorting from endosomes to the Golgi apparatus; (e) the degradation at the late endosomal/lysosomal level with formation of fragments of sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) nature; (f) the metabolic recycling of these fragments for biosynthetic purposes (salvage pathways); and (g) further degradation of fragments to waste products. Noteworthy, the correct course of ganglioside/glycosphingolipid metabolism requires the presence of the vimentin intracellular filament net work, likely to assist intracellular transport of sphingoid molecules. ut of the above events those that can be quantitatively evaluated with acceptable reliability are the processes of de novo biosynthesis, metabolic salvage and direct glycosylation. Depending on the cultured cells employed, the percentage of distribution of de novo biosynthesis, salvage pathways, and direct glycosylation, over total metabolism were reported to be: 35% (range: 10-90%) for de novo biosynthesis, 7% (range: 5-10%) for direct glycosylation, and 58% (range: 10-90%) for salvage pathways. The attempts made to calculate the half-life of overall ganglioside turnover provided data of unsure reliability, especially because in many studies salvage pathways were not taken into consideration. The values of half-life range from 2 to 6.5 h to 3 days depending on the cells used. Available evidence for changes of ganglioside/glycosphingolipid turnover, due to extracellular stimuli, is also considered and discussed.

PURINE METABOLISM IN LEUKOCYTES AND ERYTHROCYTES IN GRAVES' OR HASHIMOTO'S DISEASE

Introduction: Adenosine deaminase (ADA), purine nucleoside phosphorylase (PNPase), S-adenosylhomocysteine hydrolase (SAHH), 5′-nucleotidase (5′N), and deoxycytidine kinase (dCK) are involved in purine salvage metabolism. Changes of the activities of the above enzymes have been observed in blood cells in patients with immunological disorders. Materials and Methods: The activities of ADA, PNPase, SAHH, 5′N, and dCK in lysates of leukocytes and erythrocytes, obtained from patients with Graves' or Hashimoto's disease, were measured, using chromatographic analysis. Serum concentrations of antithyroglobulin (Tg Ab) and antithyroperoxidase (TPO Ab) antibodies were measured by an immunoenzymatic method. Results: ADA activity in leukocytes, obtained from patients with Hashimoto's disease, was significantly higher than in control leukocytes, as well as in leukocytes from patients with Graves' disease; dCK activities in leukocytes from patients with both Graves' and Hashimoto's diseases were approximately four and five times higher, respectively, than in leukocytes of control subjects; a positive correlation was observed between dCK activity in leukocytes and serum Tg Ab concentration in patients with Graves' disease. In conclusion, the increased ADA and dCK activities in leukocytes from patients with Graves' and Hashimoto's diseases may be regarded as indicators of autoimmunological thyroid diseases.

Differential Incorporation of Ara-C, Gemcitabine, and Fludarabine Into Replicating and Repairing DNA in Proliferating Human Leukemia Cells

The major actions of nucleoside analogs such as arabinosylcytosine (ara-C) and fludarabine occurs after their incorporation into DNA, during either replication or repair synthesis. The metabolic salvage and DNA incorporation of the normal nucleoside, deoxycytidine, is functionally compartmentalized toward repair synthesis in a process regulated by ribonucleotide reductase. The aim of this study was to investigate the metabolic pathways by which nucleoside analogs that do (fludarabine, gemcitabine) or do not (ara-C) affect ribonucleotide reductase are incorporated into DNA in proliferating human leukemia cells. Using alkaline density-gradient centrifugation to separate repaired DNA from replicating DNA and unreplicated parental DNA strands, approximately 60% of ara-C nucleotide in DNA was incorporated by repair synthesis in CCRF-CEM cells; the remainder was incorporated by replication. In contrast, fludarabine and gemcitabine, nucleosides that inhibit ribonucleotide reductase and decreased deoxynucleotide pools, were incorporated mainly within replicating DNA. Hydroxyurea also depleted deoxynucleotide pools and increased the incorporation of ara-C into DNA by replicative synthesis. Stimulation of DNA repair activity by UV irradiation selectively enhanced the incorporation of all nucleosides tested through repair synthesis. These findings suggest that the pathways by which therapeutically useful nucleoside analogs are incorporated into DNA are affected by cellular dNTP pools from de novo synthesis and by the relative activities of DNA repair and replication. The antitumor activity of these drugs may be enhanced by combination with either ribonucleotide reductase inhibitors to increase their incorporation into replicating DNA or with agents that induce DNA damage and evoke the DNA repair process.