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Hypoxanthine-tricyclano is a synthetic adenosine analogue, in which adenine and ribose have been replaced by hypoxanthine and a morpholino-derived tricyclic moiety, respectively. We investigated whether hypoxanthine-tricyclano could influence atrial inotropy and/or chronotropy, two important functions regulated by the A1 receptor, the main adenosine receptor type of the supraventricular myocardium. Paced left atria and spontaneously beating right atria, isolated from male, 30-35 weeks old, Wistar rats, were used. The ino- and chronotropic effects of adenosine and hypoxanthine-tricyclano (separately and together) were assessed in the absence and presence of 8-cyclopentyl-1,3-dipropylxanthine (CPX), a selective, orthosteric, reversible A1 adenosine receptor antagonist. We found that adenosine exerted a strong negative inotropic effect (similar in left and right atria). However, hypoxanthine-tricyclano elicited a moderate positive inotropic effect (also similar in all atria). In right atria, adenosine evoked a robust negative chronotropic effect, whereas hypoxanthine-tricyclano produced a slight positive chronotropy. CPX blunted the effects of both adenosine and hypoxanthine-tricyclano, although this antagonism was strong (and significant) for adenosine, while smaller (and non-significant) for hypoxanthine-tricyclano. Both effects of hypoxanthine-tricyclano were easily surmountable with adenosine. Thus, hypoxanthine-tricyclano may act as a week, orthosteric, reversible, inverse and low-affinity agonist of the A1 receptor, although alternative mechanisms of action cannot be excluded.

An effective method that can protect radiation-damaged tissues from apoptosis and promote tissue repair has not been reported to date. Hypoxanthine (Hx) is an intermediate metabolite in the purine degradation system that serves as a substrate for ATP synthesis via the salvage pathway. In this study, we focused on the transient decrease in intracellular ATP concentration after radiation exposure and examined the protective effect of Hx against radiation-induced tissue damage. Human umbilical vein endothelial cells were X irradiated, and the cell viability and incidence of apoptosis and DNA double-strand breaks (DSBs) were evaluated at different Hx concentrations. We found that in the presence of 2-100 µM Hx, the percentages of DSBs and apoptotic cells after 2, 6 and 10 Gy dose of radiation significantly decreased, whereas cell viability increased in a concentration-dependent manner. Moreover, the addition of Hx increased the levels of AMP, ADP, and ATP in the cells at 2 h postirradiation, suggesting that Hx was used for adenine nucleotide synthesis through the salvage pathway. Administration of a xanthine oxidoreductase inhibitor to a mouse model of radiation dermatitis resulted in increased blood Hx levels that inhibited severe dermatitis and accelerated recovery. In conclusion, the findings provide evidence that increasing the levels of Hx to replenish ATP could be an effective strategy to reduce radiation-induced tissue damage and elucidating the detailed mechanisms underlying the protective effects of Hx could help develop new protective strategies against radiation., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

Background: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated., Materials and Methods: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO 2 . Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex ® assay., Results: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation., Discussion: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections compels new treatment strategies, of which targeting trans-translation is promising. During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new compounds were designed. After being synthesized, the compounds were tested in vitro with saturation transfer difference (STD), fluorescence quenching titration (FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant. The active compounds provide new choices for targeting trans-translation in Mtb, and the analysis of the structure-activity relationships will be helpful for further structural modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and 2-((5-hydroxylflavone-7-yl)oxy)acetamide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887., (©AlphaMed Press 2019.)

Our laboratories have been investigating synthetic analogues of marine alkaloid rigidins that possess promising anticancer activities. These analogues, based on the 7-deazahypoxanthine skeleton, are available in one- or two-step synthetic sequences and exert cytotoxicity by disrupting microtubule dynamics in cancer cells. In the present work we extended the available structure-activity relationship (SAR) data to N3- and N9-substituted derivatives. Although N3 substitution results in loss of activity, the N9-substituted compounds retain nanomolar antiproliferative activities and the anti-tubulin mode of action of the original unsubstituted compounds. Furthermore, our results also demonstrate that multidrug-resistance (MDR) proteins do not confer resistance to both N9-unsubstituted and -substituted compounds. It was found that sublines overexpressing ABCG2, ABCC1, and ABCB1 proteins are as responsive to the rigidin analogues as their parental cell lines. Thus, the study reported herein provides further impetus to investigate the rigidin-inspired 7-deazahypoxanthines as promising anticancer agents., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lesch-Nyhan disease (LND) is a rare X-linked genetic disorder, with complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, uric acid (UA), hypoxanthine and xanthine accumulation, and a devastating neurologic syndrome. UA excess, causing renal failure, is commonly decreased by xanthine oxidoreductase (XOR) inhibitors, such as allopurinol, yielding a xanthine and hypoxanthine increase. Xanthine accumulation may result in renal stones, while hypoxanthine excess seems involved in the neurological disorder. Inhibition of purine nucleoside phosphorylase (PNP) represents a different strategy for lowering urate. PNP catalyzes the cleavage of purine ribo- and d-ribo-nucleosides into ribose/deoxyribose phosphate and free bases, starting catabolism to uric acid. Clinical trials demonstrated that PNP inhibitors, initially developed as anticancer drugs, lowered UA in some gouty patients, in association or not with allopurinol. The present study tested the reliability of an analogue of immucillin-G (C1a), a PNP inhibitor, as a therapy for urate, hypoxanthine, and xanthine excess in LND patients by blocking hypoxanthine production upstream. The therapeutic aim is to limit the administration of XOR inhibitors to LND patients by supplying the PNP inhibitor in low doses, avoiding d-nucleoside toxicity. We report studies conducted in primary cultures of skin fibroblasts from controls and LND patients grown in the presence of the PNP inhibitor. Cell viability, oxypurine release in culture medium, and endocellular nucleotide pattern have been monitored in different growth conditions (inhibitor concentration, time, added inosine). Our results demonstrate effective PNP inhibition by low inhibitor concentration, with reduced hypoxanthine release, and no appreciable toxicity in control or patient cells, suggesting a new therapeutic strategy for LND hyperuricemia., (© 2018 SSIEM.)

Hypoxanthine is the major purine involved in the salvage pathway of purines in the brain. High levels of hypoxanthine are characteristic of Lesch-Nyhan Disease. Since hypoxanthine is a purine closely related to ATP formation, the aim of this study was to investigate the effect of intrastriatal hypoxanthine administration on neuroenergetic parameters (pyruvate kinase, succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels) and mitochondrial function (mitochondrial mass and membrane potential) in striatum of rats. We also evaluated the effect of cell death parameters (necrosis and apoptosis). Wistar rats of 60 days of life underwent stereotactic surgery and were divided into two groups: control (infusion of saline 0.9%) and hypoxanthine (10 μM). Intrastriatal hypoxanthine administration did not alter pyruvate kinase activity, but increased succinate dehydrogenase and complex II activities and diminished cytochrome c oxidase activity and immunocontent. Hypoxanthine injection decreased the percentage of cells with mitochondrial membrane label and increased mitochondrial membrane potential labeling. There was a decrease in the number of live cells and an increase in the number of apoptotic cells by caused hypoxanthine. Our findings show that intrastriatal hypoxanthine administration altered neuroenergetic parameters, and caused mitochondrial dysfunction and cell death by apoptosis, suggesting that these processes may be associated, at least in part, with neurological symptoms found in patients with Lesch-Nyhan Disease.

Red cells from patients with sickle cell anemia (SCA) are under greater oxidative challenge than those from normal individuals. We postulated that oxidants generated by xanthine oxidase (XO) and hypoxanthine (HO) contribute to the pathogenesis of SCA through altering solute permeability. Sickling, activities of the main red cell dehydration pathways (P sickle , Gardos channel, and KCl cotransporter [KCC]), and cell volume were measured at 100, 30, and 0 mmHg O 2 , together with deoxygenation-induced nonelectrolyte hemolysis. Unexpectedly, XO/HO mixtures had mainly inhibitory effects on sickling, P sickle , and Gardos channel activities, while KCC activity and nonelectrolyte hemolysis were increased. Gardos channel activity was significantly elevated in red cells pharmacologically loaded with Ca 2+ using the ionophore A23187, consistent with an effect on the transport system per se as well as via Ca 2+ entry likely via the P sickle pathway. KCC activity is controlled by several pairs of conjugate protein kinases and phosphatases. Its activity, however, was also stimulated by XO/HO mixtures in red cells pretreated with N-ethylmaleimide (NEM), which is thought to prevent regulation via changes in protein phosphorylation, suggesting that the oxidants formed could also have direct effects on this transporter. In the presence of XO/HO, red cell volume was better maintained in deoxygenated red cells. Overall, the most notable effect of XO/HO mixtures was an increase in red cell fragility. These findings increase our understanding of the effects of oxidative challenge in SCA patients and are relevant to the behavior of red cells in vivo., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The mechanism of 3,5,2',4'-tetrahydroxychalcone on lowing urate level is still unknown. Here we investigated the effects of 3,5,2',4'-tetrahydroxychalcone on urate levels, xanthine oxidase/xanthine dehydrogenase (XOD/XDH) activities in hypoxanthine-induced hyperuricemic mice, as well as the effects of 3,5,2',4'-tetrahydroxychalcone on the mRNA expression levels and content of phosphoribosyl pyrophosphate synthetase (PRPS), phosphoribosyl pyrophosphate amidotransferase (PRPPAT) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Our results demonstrated that 3,5,2',4'-tetrahydroxychalcone (1.0, 2.0, and 4.0 mg/kg) reduced the uric acid levels in serum of the hyperuricemic mice in dose- and time-dependent manners. The activities of XOD/XDH in serum and liver were also significantly inhibited by 3,5,2',4'-tetrahydroxychalcone; In addition, 3,5,2',4'-tetrahydroxychalcone decreased the mRNA expression of HGPRT in brain and content of PRPS and PRPPAT in liver. These findings demonstrated that 3,5,2',4'-tetrahydroxychalcone suppresses uric acid production by affecting the critical enzymes, XOD/XDH, PRPS, PRPPAT and HGPRT in purine nucleotide metabolism.

In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, and systems biology, among others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria which induce architectural changes in biofilm colonies when cocultured with Bacillus subtilis We identified the soil bacterium Lysinibacillus fusiformis M5 as an inducer of wrinkle formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm matrix-related operons epsABCDEFGHIJKLMNO and tasA-sipW-tapA Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression but rather is caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death. IMPORTANCE Biofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be beneficial, for instance, in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they occur outside laboratory conditions. Here, we present a screening method suitable for the identification of multispecies interactions and showcase L. fusiformis as a soil bacterium that is able to live alongside B. subtilis and modify the architecture of its biofilms., (Copyright © 2017 American Society for Microbiology.)

Hypoxanthine, the major oxypurine metabolite involved in purine's salvage pathway in the brain, is accumulated in Lesch-Nyhan disease, an inborn error of metabolism of purine. The purpose of this study was to investigate the effects of hypoxanthine intrastriatal administration on infant and young adult rats submitted to stereotactic surgery. We analyzed the effect of hypoxanthine on neuroinflammatory parameters, such as cytokine levels, immunocontent of NF-κB/p65 subunit, iNOS immunocontent, nitrite levels, as well as IBA1 and GFAP immunocontent in striatum of infant and young adult rats. We also evaluate some oxidative parameters, including reactive species production, superoxide dismutase, catalase, glutathione peroxidase activities, as well as DNA damage. Wistar rats of 21 and 60 days of life underwent stereotactic surgery and were divided into two groups: control (infusion of saline 0.9 %) and hypoxanthine (10 μM). Intrastriatal administration of hypoxanthine increased IL-6 levels in striatum of both ages of rats tested, while TNF-α increased only in 21-day-old rats. Hypoxanthine also increased nuclear immunocontent of NF-κB/p65 subunit in striatum of both ages of rats. Nitrite levels were decreased in striatum of 21-day-old rats; however, the immunocontent of iNOS was increased in striatum of hypoxanthine groups. Microglial and astrocyte activation was seen by the increase in IBA1 and GFAP immunocontent, respectively, in striatum of infant rats. All oxidative parameters were altered, suggesting a strong neurotoxic hypoxanthine role on oxidative stress. According to our results, hypoxanthine intrastriatal administration increases neuroinflammatory parameters perhaps through oxidative misbalance, suggesting that this process may be involved, at least in part, to neurological disorders found in patients with Lesch-Nyhan disease.

Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)-deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H 2 O 2 also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N-acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE-siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE-deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway., (© 2016. Published by The Company of Biologists Ltd.)

Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are shown to serve as ligands for a single, critical Zn(2+) ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D "in trans", that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24×10(-5), typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3'-5' proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The antioxidant properties of Amburana cearensis extract may be a useful substitute for standard cell culture medium. Thus, the aim of this study was to evaluate the effect of this extract, with or without supplementation, on in vitro survival and development of sheep isolated secondary follicles. After collection of the ovaries, secondary follicles were isolated and cultured for 18 days in α-MEM+ supplemented with bovine serum albumin, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml. There were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml than in the control medium (α-MEM+) after 18 days of culture. Moreover, the percentage of antrum formation was significantly higher in Amb 0.1 or Amb 0.2 mg/ml than in α-MEM+ and Amb 0.1+ mg/ml, and similar to the other treatments. All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period. In conclusion, this study showed that 0.1 mg/ml of this extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. This extract can be an alternative culture medium for preantral follicle development.

Sulfur mustard (SM) is a chemical warfare agent that, upon topical application, damages skin and reaches internal organs through diffusion in blood. Two major toxic consequences of SM exposure are inflammation, associated with oxidative stress, and the formation of alkylated DNA bases. In the present study, we investigated the impact of exposure to SM on DNA repair, using two different functional DNA repair assays which provide information on several Base Excision Repair (BER) and Excision/Synthesis Repair (ESR) activities. BER activities were reduced in all organs as early as 4h after exposure, with the exception of the defense systems against 8-oxo-guanine and hypoxanthine which were stimulated. Interestingly, the resulting BER intermediates could activate inflammation signals, aggravating the inflammation triggered by SM exposure and leading to increased oxidative stress. ESR activities were found to be mostly inhibited in skin, brain and kidneys. In contrast, in the lung there was a general increase in ESR activities. In summary, exposure to SM leads to a significant decrease in DNA repair in most organs, concomitant with the formation of DNA damage. These synergistic genotoxic effects are likely to participate in the high toxicity of this alkylating agent. Lungs, possibly better equipped with repair enzymes to handle exogenous exposure, are the exception., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, reactive oxygen species (ROS) are produced by different systems which are involved in endothelial cells injury, ultimately leading to severe organ dysfunctions. The aim of this work was to study the effect of ROS produced by hypoxanthine-xanthine oxidase (Hx-XO) on the adhesion of human umbilical vein endothelial cells (HUVEC) and on the signaling pathways involved. Results show that Hx-XO-derived ROS induced an increase in HUVEC adhesion in the early stages of the process (less than 30 min), followed by a decrease in adhesion in the later stages of the process. Interestingly, Hx-XO-derived ROS induced the same biphasic effect on the phosphorylation of the focal adhesion kinase (FAK), a nonreceptor tyrosine kinase critical for cell adhesion, but not on ERK1/2 phosphorylation. The biphasic effect was not seen with ERK1/2 where a decrease in phosphorylation only was observed. Wortmannin, a PI3-kinase inhibitor, inhibited ROS-induced cell adhesion and FAK phosphorylation. Orthovanadate, a protein tyrosine phosphatase inhibitor, and Resveratrol (Resv), an antioxidant agent, protected FAK and ERK1/2 from dephosphorylation and HUVEC from ROS-induced loss of adhesion. This study shows that ROS could have both stimulatory and inhibitory effects on HUVEC adhesion and FAK phosphorylation and suggests that PI3-kinase and tyrosine phosphatase control these effects.

We studied regulation of hypoxanthine transport depending on its concentration in the culture medium. Caco-2 cells were differentiated on membrane filters to create a model of the intestine. Different hypoxanthine uptake on the apical and basolateral cell membranes was observed. The expression of SLC29 family genes encoding passive nucleoside transporters increased upon changes in hypoxanthine concentration in the medium Localization of the transporters and their influence on the effect of pharmacological preparations are discussed.

The prevalence of hyperuricemia/gout increases with aging. However, the effect of aging on function for excretion of uric acid to out of the body has not been clarified. We found that ileal uric acid clearance in middle-aged rats (11-12 months) was decreased compared with that in young rats (2 months). In middle-aged rats, xanthine oxidase (XO) activity in the ileum was significantly higher than that in young rats. Inosine-induced reactive oxygen species (ROS), which are derived from XO, also decreased ileal uric acid clearance. ROS derived from XO decreased the active homodimer level of breast cancer resistance protein (BCRP), which is a uric acid efflux transporter, in the ileum. Pre-administration of allopurinol recovered the BCRP homodimer level, resulting in the recovering ileal uric acid clearance. Moreover, we investigated the effects of ROS derived from XO on BCRP homodimer level directly in Caco-2 cells using hypoxanthine. Treatment with hypoxanthine decreased BCRP homodimer level. Treatment with hypoxanthine induced mitochondrial dysfunction, suggesting that the decreasing BCRP homodimer level might be caused by mitochondrial dysfunction. In conclusion, ROS derived from XO decrease BCRP homodimer level, resulting in suppression of function for uric acid excretion to the ileal lumen. ROS derived from XO may cause the suppression of function of the ileum for the excretion of uric acid with aging. The results of our study provide a new insight into the causes of increasing hyperuricemia/gout prevalence with aging., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5'-monophosphate (GMP) formation: conversion from xanthosine-5'-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche., (© 2015 John Wiley & Sons Ltd.)

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A series of gold(I) complexes involving triphenylphosphine (PPh3) and one N-donor ligand derived from deprotonated mono- or disubstituted hypoxanthine (HLn) of the general composition [Au(Ln)(PPh3)] (1-9) is reported. The complexes were thoroughly characterized, including multinuclear high resolution NMR spectroscopy as well as single crystal X-ray analysis (for complexes 1 and 3). The complexes were screened for their in vitro cytotoxicity against human cancer cell lines MCF7 (breast carcinoma), HOS (osteosarcoma) and THP-1 (monocytic leukaemia), which identified the complexes 4-6 as the most promising representatives, who antiproliferative activity was further tested against A549 (lung adenocarcinoma), G-361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) cell lines. Complexes 4-6 showed a significantly higher in vitro anticancer effect against the employed cancer cells, except for G-361, as compared with the commercially used anticancer drug cisplatin, with IC50 ≈ 1-30 µM. Anti-inflammatory activity was evaluated in vitro by the assessment of the ability of the complexes to modulate secretion of the pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the lipopolysaccharide-activated macrophage-like THP-1 cell model. The results of this study identified the complexes as auspicious anti-inflammatory agents with similar or better activity as compared with the clinically applied gold-based antiarthritic drug Auranofin. In an effort to explore the possible mechanisms responsible for the biological effect, the products of interactions of selected complexes with sulfur-containing biomolecules (L-cysteine and reduced glutathione) were studied by means of the mass-spectrometry study.

In the present study, we investigated the in vitro effect of hypoxanthine on the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase, as well as on thiobarbituric-acid-reactive substances (TBA-RS), in the renal cortex and medulla of rats. Results showed that hypoxanthine, at a concentration of 10.0 μM, enhanced the activities of CAT and SOD in the renal cortex of 15-, 30- and 60-day-old rats, enhanced SOD activity in the renal medulla of 60-day-old rats and enhanced TBA-RS levels in the renal medulla of 30-day-old rats, as compared with controls. Furthermore, we also verified the influence of allopurinol (an inhibitor of xanthine oxidase), as well as of the antioxidants, trolox and ascorbic acid on the effects elicited by hypoxanthine on the parameters tested. Allopurinol and/or administration of antioxidants prevented most alterations caused by hypoxanthine in the oxidative stress parameters evaluated. Data suggest that hypoxanthine alters antioxidant defences and induces lipid peroxidation in the kidney of rats; however, in the presence of allopurinol and antioxidants, some of these alterations in oxidative stress were prevented. Our findings lend support to a potential therapeutic strategy for this condition, which may include the use of appropriate antioxidants for ameliorating the damage caused by hypoxanthine., (Copyright © 2014 John Wiley & Sons, Ltd.)

Arthrobacter sp. CGMCC 3584 are able to produce cAMP from glucose by the purine synthesis pathway via de novo or salvage biosynthesis. In order to gain an improved understanding of its metabolism, (13)C-labeling experiment and gas chromatography-mass spectrometry (GC-MS) analysis were employed to determine the metabolic network structure and estimate the intracellular fluxes. GC-MS analysis helps to reflect the activity of the intracellular pathways and reactions. The metabolic network mainly contains glycolytic and pentose phosphate pathways, the tricarboxylic acid cycle, and the inactive glyoxylate shunt. Hypoxanthine as a precursor of cAMP and sodium fluoride as an inhibitor of glycolysis were found to increase the cAMP production, as well as the flux through the PP pathway. The effects of adding hypoxanthine and sodium fluoride are discussed based on the enzyme assays and metabolic flux analysis. In conclusion, our results provide quantitative insights into how cells manipulate the metabolic network under different culture conditions and this may be of value in metabolic regulation for desirable production., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The structure and composition of the endothelial cell (EC) glycocalyx reflect a balance of the biosynthesis of glycans and their shear dependent removal. Shedding of glycans from the EC surface has been shown to occur in response to reactive oxygen species (ROS) and inflammatory mediators. Using sub-antimicrobial doses of doxycycline, a broad spectrum matrix metalloprotease (MMP) inhibitor, inhibition of chemoattractant induced glycan shedding has suggested that MMPs may be a major effector of the loss of glycans. However, it has also been reported that doxycycline is a scavenger of ROS that may also activate MMPs. To clarify the basis for doxycycline as an inhibitor of glycan shedding, the present studies were undertaken to determine its effect on ROS induced shedding in post-capillary venules of the exteriorized mesentery of the rat. To this end, hypoxanthine (HX) and xanthine oxidase (XO) were rapidly mixed on the mesenteric surface for a 2min period to generate superoxide anion (O2(-)·) and the time course of glycan shedding was monitored in post-capillary venules over a 30min period. Glycan shedding was quantitated by loss of adherent fluorescently labeled lectin coated microspheres (FLMs, 0.1μm diameter) that were systemically infused. It was found that HX/XO caused FLM adhesion to decrease 45% within 30min. This effect could be inhibited in a dose dependent manner by the addition of superoxide dismutase to the superfusion solution, thus confirming the role of O2(-)·. In contrast, 0.5μM doxycycline had no effect on FLM shedding in response to HX/XO, contrary to its ability to attenuate shedding in response to the chemoattractant fMLP. Thus it is suggested that the efficacy of doxycycline as an inhibitor of glycan shedding during inflammation arises from its ability to inhibit MMP activation., (© 2013.)

In the present study, we investigate the in vitro effect of hypoxanthine on acetylcholinesterase and butyrylcholinesterase activities in the hippocampus, striatum, cerebral cortex and serum of 15-, 30- and 60-day-old rats. Furthermore, we also evaluated the influence of antioxidants, namely α-tocopherol (trolox) and ascorbic acid, and allopurinol to investigate the possible participation of free radicals and uric acid in the effects elicited by hypoxanthine on these parameters. Acetylcholinesterase and butyrylcholinesterase activities were determined according to Ellman et al. (Biochem Pharmacol 7:88-95, 1961), with some modifications. Hypoxanthine (10.0 μM), when added to the incubation medium, enhanced acetylcholinesterase activity in the hippocampus and striatum of 15- and 30-day-old rats and reduced butyrylcholinesterase activity in the serum of 60-day-old rats. The administration of allopurinol and/or antioxidants partially prevented the alterations caused by hypoxanthine in acetylcholinesterase and butyrylcholinesterase activities in the cerebrum and serum of rats. Data indicate that hypoxanthine alters cholinesterase activities, probably through free radicals and uric acid production since the alterations were prevented by the administration of allopurinol and antioxidants. It is presumed that the cholinesterase system may be associated, at least in part, with the neuronal dysfunction observed in patients affected by Lesch-Nyhan disease. In addition, although extrapolation of findings from animal experiments to humans is difficult, it is conceivable that these vitamins and allopurinol might serve as an adjuvant therapy to avoid progression of brain damage in patients affected by this disease.

The water-soluble B vitamins, folate and folic acid, play an important role in reproductive health, but little is known about the effects of folic acid on infertility. The present study tested the hypothesis that folic acid affects oocyte maturation, a possible cause of female infertility. We have studied the in vitro maturation of mouse and Xenopus oocytes. Hypoxanthine (Hx) was used as an inhibitor of mouse oocyte maturation to mimic in vivo conditions by maintaining high levels of cyclic-AMP. The frequency of first polar body (PB1) formation and germinal vesicle breakdown (GVBD) in mouse oocytes was decreased by Hx. This effect was counteracted by folic acid added to the medium. PB1 extrusion and GVBD percentages rose to 27·7 and 40·0% from 12·8 and 19·9%, respectively, by exposure to 500 μM-folic acid. Folic acid also restored the spindle configuration, which had been elongated by Hx, as well as normalising the distribution of cortical granules (CG). In folic acid-treated Xenopus eggs, extracellular signal-regulated kinase 1 was phosphorylated, cyclin B2 and Mos were up-regulated and the frequency of GVBD was accelerated. Taken together, the findings suggest that folic acid facilitates oocyte maturation by altering the expression and phosphorylation of proteins involved in M-phase-promoting factor and mitogen-activated protein kinase pathways, as well as causing changes in spindle configuration and CG migration.

Oxidative stress has been implicated in pathogenesis of many diseases, but few studies describe its influence on spermatogenesis. In this study, we analyzed the direct influence of hypoxanthine (Hx)-induced reactive oxygen species (ROS) on spermatogenesis in fish using the Japanese eel (Anguilla japonica) testicular organ culture system. Testicular fragments of eels were cultured in 0.1-100 μM Hx with or without 10 ng/ml 11-ketotestosterone (11-KT). Immunohistochemistry for 5-bromo-2-deoxyuridine showed that Hx treatment at a low dose (1 μM) already inhibits 11-KT-induced germ cell proliferation after culture. An in situ TUNEL assay and 8-hydroxy-2'-deoxyguanosine immunohistochemistry revealed an intense germ cell apoptosis and high oxidative DNA damage in testicular fragments cultured at the highest dose of Hx (100 μM) with 11-KT. A total superoxide dismutase (SOD) activity assay showed a decrease in SOD activity in testicular fragments cultured with 11-KT. These data suggest that ROS may directly inhibit spermatogenesis, and that decreased SOD activity renders proliferating spermatogonia susceptible to ROS, hence leading to apoptosis.

In our previous study, we demonstrated that combinatorial addition of hypoxanthine (10 mg/L) and thymidine (2 mg/L) was able to stimulate initial cell growth and elevate volumetric concentration of antibody by 22% (Chen et al., Appl. Microbiol. Biotechnol., 93, 169-178, 2012). In this study, a systematic study was carried out to investigate the effects of hypoxanthine and thymidine (H&T) on cell growth and antibody production in a much wider range of concentration. In addition, we pursued to establish a highly productive fed-batch culture via rationally designing H&T addition regime. It was found that both cell growth and antibody production in batch cultures were H&T concentration-dependent. Specifically, a low concentration stimulated cell growth while exerting no influence on specific productivity (q(mAb)), and a high concentration inhibited cell growth, however, significantly enhancing q(mAb). Subsequent experiments with fed-batch shaking flasks demonstrated the feasibility of improving antibody production using a biphasic addition strategy for H&T: supplementing a low concentration of H&T during initial cell growth phase and a high concentration of H&T at the production phase. By applying the optimized feeding regime, a maximum viable cell density (VCD) of 6.45 × 10(6)cells/mL and volumetric antibody production of 632 mg/L were achieved in a 2 L-B.Braun bioreactor. Taken together, in this study, a biphasic H&T addition strategy for cell culture was developed, which hold great promise to improve antibody production., (Copyright © 2012. Published by Elsevier B.V.)

Serine racemase (SR) is the only identified enzyme in mammals responsible for isomerization of L-serine to D-serine, a coagonist at N-methyl-D-aspartate (NMDA) receptors in the forebrain. Our previous data showed that an apparent SR dimer resistant to sodium dodecyl sulfate and β-mercaptoethanol was elevated in microglial cells after proinflammatory activation. Because the activation of microglia is typically associated with an oxidative burst, oxidative cross-linking between SR subunits was speculated. In this study, an siRNA technique was employed to confirm the identity of this SR dimer band. The oxidative species potentially responsible for the cross-linking was investigated with recombinant SR protein. The data indicate that nitric oxide, peroxynitrite, and hydroxyl radical were the likely candidates, whereas superoxide and hydrogen peroxide per se failed to contribute. Furthermore, the mechanism of formation of SR dimer by peroxynitrite oxidation was studied by mass spectrometry. A disulfide bond between Cys₆ and Cys₁₁₃ was identified in 3-morpholinosydnonimine hydrochloride (SIN-1)-treated SR monomer and dimer. Activity assays indicated that SIN-1 treatment decreased SR activity, confirming our previous conclusion that noncovalent dimer is the most active form of SR. These findings suggest a compensatory feedback in which the consequences of neuroinflammation might dampen D-serine production to limit excitotoxic stimulation of NMDA receptors., (Copyright © 2012 Wiley Periodicals, Inc.)

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.

Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO), or dissected follicles were obtained 44-48 hr after priming immature mice (20-23 days old) with 5 IU or immature rats (25-27 days old) with 12.5 IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. Atrial natriuretic peptide, a guanylate cyclase activator, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to undergo germinal vesicle breakdown (GVB) by follicle-stimulating hormone (FSH) and amphiregulin, while human chorionic gonadotropin (hCG) was ineffective in both species. Nevertheless, FSH and amphiregulin stimulated cumulus expansion in both species. FSH and hCG were both effective inducers of GVB in cultured mouse and rat follicles while amphiregulin was stimulatory only in mouse follicles. Changing the culture medium or altering macromolecular supplementation had no effect on FSH-induced maturation in rat CEO. The AMP-activated protein kinase (AMPK) activator, AICAR, was a potent stimulator of maturation in mouse CEO and DO, but only marginally stimulatory in rat CEO and ineffective in rat DO. The AMPK inhibitor, compound C, blocked meiotic induction more effectively in hCG-treated mouse follicles and heat-treated mouse CEO. Both agents produced contrasting results on polar body formation in cultured CEO in the two species. Active AMPK was detected in germinal vesicles of immature mouse, but not rat, oocytes prior to hCG-induced maturation in vivo; it colocalized with chromatin after GVB in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. Finally, cultured mouse and rat CEO displayed disparate maturation responses to energy substrate manipulation. These data highlight significant differences in meiotic regulation between the two species, and demonstrate a greater potential in mice for control at the level of the cumulus CEO., (Copyright © 2011 Wiley Periodicals, Inc.)

Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKCα and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis., (Copyright © 2011. Published by Elsevier Inc.)

DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown., Methodology/principal Findings: Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage., Conclusions/significance: These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.

The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging.

Purpose: Nucleoside and base modulation of the cytotoxicity of nucleic acid and folate antimetabolite drugs has been widely discussed. Many investigators have observed reduced toxicity due to circumvention of drug-induced inhibition of de novo purine and pyrimidine synthesis. However, exogenous purine nucleosides and bases may also enhance the cytotoxicity of even moderate concentrations of antifolate drugs (MTX and PTX) which inhibit dihydrofolate reductase. In this study, the effects of nucleosides in the medium on the cytotoxicity and deoxyribonucleoside triphosphate pools after brief exposure of cultured cells to methotrexate have been studied in cultured L1210 murine leukaemia cells., Methods: Cell viability was determined by trypan blue exclusion assay. Colony formation was assessed by microtitration cloning assay. The deoxyribonucleotides were measured by a modification of the DNA polymerase assay. Purines were extracted with trioctylamine and 1,1,2-trichlorotrifluoroethane buffer and concentrations of purine bases were determined by HPLC., Results: Subculture of drug-treated cells in fresh medium containing 10% FCS led to greater toxicity than sub culture in 'conditioned' medium, i.e. fresh medium in which logarithmically growing cells had been cultured for 24 h before separation. Cells resuspended in fresh medium had increased dATP and sustained inhibition of dTTP levels, while cells subcultured in 'conditioned' medium had no elevation of dATP. Hypoxanthine concentration determined by HPLC in 'conditioned' medium was 0.9 microM compared to 6.7 microM in fresh medium. Resuspension of drug-treated cells in conditioned medium supplemented with 10 or 100 microM HX enhanced cytotoxicity and increased the dATP levels., Conclusion: These results add further evidence that purines present in normal culture conditions are important determinants of methotrexate cytotoxicity. Elevation of dATP levels after methotrexate treatment is an important modulator of cytotoxicity.

To improve developmental competence of in vitro matured oocytes, culture medium can be supplemented with hypoxanthine (Hx) and FSH or epidermal growth factor (EGF) to trigger the activation of essential signalling pathways regulating meiotic resumption and progression. Since the serine/threonine kinase, Akt, contributes to the regulation of the meiotic cell cycle, this study analysed its expression level and localization at the meiotic spindle in oocytes matured in vivo or in vitro in the presence of Hx-FSH or Hx-EGF. Independently of culture conditions adopted, Akt mRNA concentration did not vary from germinal vesicle to metaphase I (MI), while at MII a significant decrease in Akt1 mRNA concentration was recorded in oocytes matured in vivo and in those stimulated by Hx-EGF (P < 0.05). Phoshorylated Akt protein content was similar in the different groups of MI oocytes, but it decreased at MII in oocytes matured either in vivo or in vitro with Hx-EGF. Ser-473-phosphorylated Akt was localized uniformly to the meiotic spindle in more than 90% of oocytes. These results indicate that, in mouse oocytes, Akt expression is differentially regulated during in vivo and in vitro maturation and suggest that EGF could be a positive modulator, even stronger than FSH, of oocyte meiotic maturation., (Copyright (c) 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Self-injurious behavior is the most outstanding feature of Lesch-Nyhan syndrome and has recently been ascribed to an obsessive-compulsive behavior. Lesch-Nyhan syndrome results from the complete enzyme deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) but the link between abnormal purine metabolism and its neurological and behavioral manifestations remains largely unknown. Previous studies led us to hypothesize that adenosine and dopamine receptor expression could be altered in HPRT-deficient cells. To test this hypothesis, we examined mRNA expressions of adenosine (ADORA2A and ADORA2B) and dopamine receptors (DRD1 and DRD2 like), and dopamine transporter (DAT1) in peripheral blood lymphocytes (PBLs) from Lesch-Nyhan patients. We also examined the influence of hypoxanthine in these expressions. As compared to normal PBLs, both ADORA2A and DRD5 expression were abnormal in PBLs from Lesch-Nyhan patients. In contrast, DAT1 expression was similar to control values in HPRT deficient PBLs. These results indicate an abnormal adenosine and dopamine receptor expression in HPRT-deficient cells and suggest disrupted adenosine and dopamine neurotransmission may have a significant role in the pathogenesis of the neurological manifestations of Lesch-Nyhan syndrome.

Background: Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still unsatisfying. New treatment strategies have to be developed., Materials and Methods: A hypoxanthine/aminopterin/thymidine (HAT)-sensitive EFT cell line was developed by repetitive treatment of the EFT cell line SK-N-MC with 8'-azaguanine (8AG). By using DNA microarrays, the gene expression profile of this cell line was characterized. Immunostimulatory activity was assessed by mixed lymphocyte/tumour cell culture (MLTC). Artificial fusion of tumour cells and dendritic cells was visualized by flow cytometry., Results: After selection of 8AG-resistant cells, a cell line with high sensitivity for treatment with HAT was obtained. Expression of the X chromosome inactivation specific transcript XIST was higher in HAT-sensitive cells. Nevertheless, HAT-sensitive cells retained the EFT-associated gene expression profile. Moreover, in the presence of HAT, it was possible to use these cells without irradiation as stimulatory cells in MLTC or as fusion partner for dendritic cells., Conclusion: HAT-sensitive EFT cells might be an interesting tool for the development of new immunotherapeutic approaches for the treatment of EFT.

In prokaryotes, menaquinone is used for respiration. In Escherichia coli, menaquinone is biosynthesized from chorismate by seven enzymes. However, very recently, we identified an alternative pathway (the futalosine pathway), which operates in some bacteria, including Streptomyces coelicolor, Helicobacter pylori, Campylobacter jejuni, and Thermus thermophilus. We describe the steps of this pathway, which branches at chorismate in a manner similar to the known pathway, but then follows a different route. This new pathway includes futalosine, an unusual nucleoside derivative consisting of inosine and o-substituted benzoate moieties, as a biosynthetic intermediate. In this study, a recombinant futalosine hydrolase (TTHA0556) of T. thermophilus, which participates in the second step of the pathway and catalyzes the reaction releasing hypoxanthine from futalosine, was prepared and used in functional analyses. Recombinant TTHA0556 formed a homotetramer and reacted only with futalosine; other structurally related nucleotides and nucleosides were not accepted. Recombinant TTHA0556 required no cofactors, and the optimum pH and temperature were 4.5 and 80 degrees C. The Km value was calculated to be 154.0+/-5.3 microM and the kcat value was 1.02/s. Recombinant TTHA0556 was slightly inhibited by hypoxanthine, with a Ki value of 1.1 mM.

New polymethylene derivatives of nucleic bases with a beta-diketo function in the omega-position were obtained by alkylation of uracil, thymine, cytosine, hypoxanthine, adenine, and N(2)-isobutyryl guanine with 2-omega-chloroal-kanoyl)cyclohexanones. The physical and chemical characteristics of the compounds synthesized and their effect on the K562 and HCT116 tumor cell lines were studied.

The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine on ectonucleotidase (E-NTPDases and ecto-5'-nucleotidase) activities and expressions in the striatum of rats. The effect of pre-treatment with vitamins E and C on the effects elicited by this oxypurine on enzymatic activities and on thiobarbituric reactive substances (TBARS) was also investigated. The effect of pre-incubation with hypoxanthine on nucleotide hydrolysis in striatum homogenate was also determined. Adult Wistar rats were divided into (1) control and (2) hypoxanthine-injected groups. For ectonucleotidase activity determination, the animals were sacrificed at 30 min, 24 h and 7 days after drug infusion. For the evaluation of the expression of NTPDase 1-3 and also ecto-5'-nucleotidase, TBARS assay and the influence of the pre-treatment with vitamins on ectonucleotidase activities, the animals were sacrificed 24 h after hypoxanthine infusion. Results show that hypoxanthine infusion significantly inhibited ectonucleotidase activities and increased TBARS only 24 h after administration. Pre-treatment with vitamins was able to prevent these effects. Moreover, ecto-5'-nucleotidase expression was increased (80%) at 24 h after hypoxanthine infusion. We suggest that these hypoxanthine-induced biochemical modifications could, at least in part, participate in the pathophysiology of Lesch Nyhan disease.

The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine, the major compound accumulated in Lesch-Nyhan disease, on performance step-down inhibitory avoidance task in the rat. Male adult Wistar rats were divided in two groups: (1) saline-injected and (2) hypoxanthine-injected group. Treated-group received intrastriatal hypoxanthine solution 30 min before training session (memory acquisition) or immediately after training session (memory consolidation) or 30 before test session (memory retrieval) on step-down inhibitory avoidance task. Results show that hypoxanthine administration caused significant memory impairment in all periods tested. These results show that intrastriatal hypoxanthine administration provoked memory process impairment of step-down inhibitory avoidance task, an effect that might be related to the cognitive memory alterations in Lesch-Nyhan patients.

We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.

Background: 5-Fluorouracil (5-FU) is an antineoplastic drug that targets thymidylate synthase (TS). Tumour cells can develop resistance to anti-TS drugs by a variety of mechanisms including up-regulation of TS protein and alterations in drug uptake and degradation. The possible mechanisms of the observed rapid development of resistance to the pyrimidine analogs 5-FUdR and 5-FU in cultured HCT116 colon cancer cells were investigated., Materials and Methods: Cell survival was determined in resistant and control HCT116 cells treated with 5-FUdR and 5-FU for 7 days. The ability of the cells to take up and metabolize these drugs was determined by Western blotting and [3H]thymidine incorporation., Results and Conclusion: Resistant HCT116 cells were 5- and 100-fold more resistant to killing by 5-FU and 5-FUdR, respectively, than the parental cells and exhibited impaired uptake. Although the HCT116R cells were initially Mycoplasma free, a low level of Mycoplasma contamination was found in these cells after several weeks in culture. Sensitivity to 5-FUdR was restored by treatment with an anti-Mycoplasma antibiotic. Our observations emphasize the need for frequent testing for Mycoplasma contamination in any cell line under investigation for resistance to anti-TS drugs.

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 micromol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-l-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 micromol/l had no effect on ATP concentrations and did not increase the activities of either the alpha1 or alpha2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism.

Purines are synthesized de novo in 10 chemical steps that are catalyzed by six enzymes in eukaryotes. Studies in vitro have provided little evidence of anticipated protein-protein interactions that would enable substrate channeling and regulation of the metabolic flux. We applied fluorescence microscopy to HeLa cells and discovered that all six enzymes colocalize to form clusters in the cellular cytoplasm. The association and dissociation of these enzyme clusters can be regulated dynamically, by either changing the purine levels of or adding exogenous agents to the culture media. Collectively, the data provide strong evidence for the formation of a multi-enzyme complex, the "purinosome," to carry out de novo purine biosynthesis in cells.

The heart responds to an increased demand arising due to physiological stimuli or pathological insults by hypertrophy of myocytes. Reactive oxygen species (ROS) have recently been identified as the molecular intermediates in the translation of mechanical stimuli to cellular response. Different signal transduction pathways have been implicated with cardiac hypertrophy, prominent among them being, mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and calcineurin. It remains unclear whether the ROS induced hypertrophy is mediated through one or more of these pathways. This study was taken up with the objective to affirm the role of ROS in the induction of cardiomyocyte hypertrophy and examine the contribution of specific pathways in the mediation of the hypertrophic response. The cellular response to enzyme-generated reactive oxygen species was examined in cultured cells from newborn rat heart. Pathway specific inhibitors were used to identify the role of each pathway in the mediation of cellular hypertrophy. Cellular hypertrophy in response to hypoxanthine-xanthine oxidase was prevented by inhibition of any one of the pathways; leading to the inference that oxidative stress induced hypertrophy is mediated by coordinative regulation of the three major pathways.