Expression and purification of the 5'-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology.

5'-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides to their corresponding nucleosides plus phosphate. In the present study, to search for new genes encoding 5'-nucleotidases specific for purine nucleotides in industrially important Bacillus species, "shotgun" cloning and the direct selection of recombinant clones grown in purine nucleosides at inhibitory concentrations were performed in the Escherichia coli GS72 strain, which is sensitive to these compounds. As a result, orthologous yitU genes from Bacillus subtilis and Bacillus amyloliquefaciens, whose products belong to the ubiquitous haloacid dehalogenase superfamily (HADSF), were selected and found to have a high sequence similarity of 87%. B. subtilis YitU was produced in E. coli as an N-terminal hexahistidine-tagged protein, purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity with respect to various deoxyribo- and ribonucleoside monophosphates: dAMP, GMP, dGMP, CMP, AMP, XMP, IMP and 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR-P). However, the preferred substrate for recombinant YitU was shown to be flavin mononucleotide (FMN). B. subtilis and B. amyloliquefaciens yitU overexpression increased riboflavin (RF) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) accumulation and can be applied to breed highly performing RF- and AICAR-producing strains.

Purine nucleotide metabolism regulates expression of the human immune ligand MICA.

Expression of the cell-surface glycoprotein MHC class I polypeptide-related sequence A (MICA) is induced in dangerous, abnormal, or "stressed" cells, including cancer cells, virus-infected cells, and rapidly proliferating cells. MICA is recognized by the activating immune cell receptor natural killer group 2D (NKG2D), providing a mechanism by which immune cells can identify and potentially eliminate pathological cells. Immune recognition through NKG2D is implicated in cancer, atherosclerosis, transplant rejection, and inflammatory diseases, such as rheumatoid arthritis. Despite the wide range of potential therapeutic applications of MICA manipulation, the factors that control MICA expression are unclear. Here we use metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a "Warburg" or biosynthetic metabolic state induces MICA expression. Specifically, we show that glucose transport into the cell and active glycolytic metabolism are necessary to up-regulate MICA expression. Active purine synthesis is necessary to support this effect of glucose, and increases in purine nucleotide levels are sufficient to induce MICA expression. Metabolic induction of MICA expression directly influences NKG2D-dependent cytotoxicity by immune cells. These findings support a model of MICA regulation whereby the purine metabolic activity of individual cells is reflected by cell-surface MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.

P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors.

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation.

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3+T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2-200µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5'-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

Synthesis and biological profiling of 6- or 7-(het)aryl-7-deazapurine 4'-C-methylribonucleosides.

The synthesis and biological activity profiling of a large series of diverse pyrrolo[2,3-d]pyrimidine 4'-C-methylribonucleosides bearing an (het)aryl group at position 4 or 5 is reported as well as the synthesis of several phosphoramidate prodrugs. These compounds are 4'-C-methyl derivatives of previously reported cytostatic hetaryl-7-deazapurine ribonucleosides. The synthesis is based on glycosylation of halogenated 7-deazapurine bases with 1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-methyl-ß-d-ribofuranose followed by cross-coupling and nucleophilic substitution reactions. The final compounds showed low cytotoxicity and several derivatives exerted antiviral activity against HCV or Dengue viruses at micromolar concentrations.

Stability and immunogenicity properties of the gene-silencing polypurine reverse Hoogsteen hairpins.

Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-κB were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-α, IFN-α, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.

Ubiquinol (QH(2)) functions as a negative regulator of purine nucleotide inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein.

We compared the influence of different adenine and guanine nucleotides on the free fatty acid-induced uncoupling protein (UCP) activity in non-phosphorylating Acanthamoeba castellanii mitochondria when the membranous ubiquinone (Q) redox state was varied. The purine nucleotides exhibit an inhibitory effect in the following descending order: GTP>ATP>GDP>ADP≫GMP>AMP. The efficiency of guanine and adenine nucleotides to inhibit UCP-sustained uncoupling in A. castellanii mitochondria depends on the Q redox state. Inhibition by purine nucleotides can be increased with decreasing Q reduction level (thereby ubiquinol, QH2 concentration) even with nucleoside monophosphates that are very weak inhibitors at the initial respiration. On the other hand, the inhibition can be alleviated with increasing Q reduction level (thereby QH2 concentration). The most important finding was that ubiquinol (QH2) but not oxidised Q functions as a negative regulator of UCP inhibition by purine nucleotides. For a given concentration of QH2, the linoleic acid-induced GTP-inhibited H(+) leak was the same for two types of A. castellanii mitochondria that differ in the endogenous Q content. When availability of the inhibitor (GTP) or the negative inhibition modulator (QH2) was changed, a competitive influence on the UCP activity was observed. QH2 decreases the affinity of UCP for GTP and, vice versa, GTP decreases the affinity of UCP for QH2. These results describe the kinetic mechanism of regulation of UCP affinity for purine nucleotides by endogenous QH2 in the mitochondria of a unicellular eukaryote.

High-energy compounds promote physiological processing of Alzheimer's amyloid-ß precursor protein and boost cell survival in culture.

Physiological or α-processing of amyloid-ß precursor protein (APP) prevents the formation of Aß, which is deposited in the aging brain and may contribute to Alzheimer's disease. As such, drugs promoting this pathway could be useful for prevention of the disease. Along this line, we searched through a number of substances and unexpectedly found that a group of high-energy compounds (HECs), namely ATP, phosphocreatine, and acetyl coenzyme A, potently increased APP α-processing in cultured SH-SY5Y cells, whereas their cognate counterparts, i.e., ADP, creatine, or coenzyme A did not show the same effects. Other HECs such as GTP, CTP, phosphoenol pyruvate, and S-adenosylmethionine also promoted APP α-processing with varying potencies and the effects were abolished by energy inhibitors rotenone or NaN(3). The overall efficacy of the HECs in the process ranged from three- to four-fold, which was significantly greater than that exhibited by other physiological stimulators such as glutamate and nicotine. This suggested that the HECs were perhaps the most efficient physiological stimulators for APP α-processing. Moreover, the HECs largely offset the inefficient APP α-processing in aged human fibroblasts or in cells impaired by rotenone or H(2) O(2). Most importantly, some HECs markedly boosted the survival rate of SH-SY5Y cells in the death process induced by energy suppression or oxidative stress. These findings suggest a new, energy-dependent regulatory mechanism for the putative α-secretase and thus will help substantially in its identification. At the same time, the study raises the possibility that the HECs may be useful to energize and strengthen the aging brain cells to slow down the progression of Alzheimer's disease.

[Effects of purine nucleotide on the expressions of FSH and LH and the ultrastructure of endocrine cells in the pituitary gland of heroin-addicted male rats].

OBJECTIVE: To investigate the effects of purine nucleotide on the expressions of follicle-stimulating hormone (FSH) and luteotrophic hormone (LH) and the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of heroin-addicted and -withdrawal rats. METHODS: Ninety-two male Wistar rats were randomly divided into a control group (ip saline for 14 d), a nucleotide group (ip AMP and GMP for 10 d), a heroin group (ip heroin for 10 d), a heroin + nucleotide group (ip AMP and GMP + heroin for 10 d), a 3 d withdrawal group (ip heroin for 10 d and killed at 14 d), a 9 d withdrawal group (ip heroin for 10 d and killed at 20 d), a 3 d nucleotide group (ip nucleotide for 3 d after 10 d heroin administration and killed at 14 d), and a 9 d nucleotide group (ip nucleotide for 9 d after 10 d heroin administration and killed at 20 d). Changes in the mRNA expressions of FSH and LH in the pituitary gland of the rats were analyzed by semi-quantitative RT-PCR, and alterations in the ultrastructures of the distal somatotrophic and gonadotrophic cells were observed under the microscope. RESULTS: The expression of FSH mRNA was significantly increased in the nucleotide, heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.099 +/- 0.018, 0.177 +/- 0.046, 0.151 +/- 0.030 and 0.184 +/- 0.028) as compared with the control group (0.045 +/- 0.009) (P < 0.01); and so was that of LH mRNA in the heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.950 +/- 0.169, 0.990 +/- 0.171 and 0.960 +/- 0.147) in comparison with the control group (0.700 +/- 0.099) (P < 0.01). In the heroin group, the nuclei of the distal somatotrophic and gonadotrophic cells exhibited morphological abnormality, unclear membrane, slightly pyknotic matrix, marginal and agglutinated heterochromatin, dilated rough endoplasmic reticula, swollen mitochondria, broken and vacuolated cristae in the cytoplasm, obviously decreased number of secretory granules, and myelin bodies in some cells. However, the heroin + nucleotide group showed no significant changes in the ultrastructures of somatotrophic and gonadotrophic cells compared with the control group. CONCLUSION: Short-term use of heroin does not obviously affect the expressions of FSH and LH mRNA in the pituitary gland of rats, while heroin + nucleotide, or nucleotide following heroin withdrawal can enhance their expressions significantly. Heroin damages the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of male rats, and purine nucleotide can diminish or inhibit this damage.

Metformin alleviates long-term high-fructose diet-induced skeletal muscle insulin resistance in rats by regulating purine nucleotide cycle.

Nutrient excess caused by excessive fructose intake can lead to insulin resistance and dyslipidemia, which further causes the development of metabolic syndrome. Metformin is a well-known AMPK activator widely used for the treatment of metabolic syndrome, while the mechanism of AMPK activation remains unclear. The present study aimed to investigate the pharmacological effects of metformin on fructose-induced insulin resistance rat, and the potential mechanism underlying AMPK activation in skeletal muscle tissue. Results indicated that metformin significantly ameliorated features of insulin resistance, including body weight, Lee's index, hyperinsulinemia, dyslipidemia, insulin intolerance and pancreatic damage. Moreover, treatment with metformin attenuated the inflammatory response in serum and enhanced the antioxidant capacity in skeletal muscle tissue. The therapeutic effects of metformin on fructose-induced insulin resistance may be related to the activation of AMPK to regulate Nrf2 pathway and mitochondrial abnormality. Additionally, metformin suppressed the expression of adenosine monophosphate deaminase 1 (AMPD1) and up-regulated the expression of adenylosuccinate synthetase (ADSS) in the purine nucleotide cycle (PNC), which facilitated the increase of AMP level and the ratio of AMP/ATP. Therefore, we proposed a novel mechanism that metformin activated AMPK via increasing AMP by regulating the expression of AMPD1 and ADSS in PNC pathway.

Mitochondrial uncoupling proteins in unicellular eukaryotes.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.

Insights into the cellular pharmacokinetics and pharmacodynamics of thiopurine antimetabolites in a model of human intestinal cells.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity.

Synthesis of cyclic di-nucleotidic acids as potential inhibitors targeting diguanylate cyclase.

Five analogs of cyclic di-nucleotidic acid including c-di-GMP were synthesized and evaluated for their biological activities on Slr1143, a diguanylate cyclase of Synechocystis sp. Slr1143 was overexpressed from the recombinant plasmid which contained the gene of interest and subsequently purified by affinity chromatography. A new HPLC method capable of separating the compound and product peaks with good resolution was optimized and applied to the analysis of the compounds. Results obtained show that cyclic di-inosinylic acid 1b demonstrates a stronger inhibition on Slr1143 than c-di-GMP and is a potential inhibitor for biofilm formation.

Behavioral response of human spermatozoa to a concentration jump of chemoattractants or intracellular cyclic nucleotides.

BACKGROUND: A major question in mammalian sperm chemotaxis is whether the cells sense a chemoattractant gradient by comparing the chemoattractant concentration between time points or between spatial points. METHODS: To resolve this question, we exposed human spermatozoa to a temporal chemoattractant gradient under conditions of no spatial gradient by rapidly mixing the cells with progesterone or bourgeonal on a microscope slide and analyzing their swimming with motion analysis software. RESULTS: The cells responded within seconds with an increase in velocity and lateral head displacement, and with a decrease in the linearity of swimming, becoming hyperactivated at the peak of the response. All the responses were transient, lasting for a number of seconds. Essentially similar results were obtained upon intracellular photorelease of cyclic adenosine monophosphate or cyclic guanosine monophosphate, which are thought to be involved in mediating the chemotactic response. CONCLUSION: These results suggest that human spermatozoa sense and respond to a temporal chemoattractant gradient. On the basis of these observations, we propose a potential model for the chemotactic response of spermatozoa in a spatial chemoattractant gradient.

Bone cells in culture: morphologic transformation by hormones.

Hormones and purine nucleosides and nucleotides induced cultured bone cells to transform transiently from a spherical to a stellate shape. Cytochalasin B also induced the transformation. The change was blocked by colchicine and vinblastine, but not by lumicolchicine or cycloheximide. This morphologic transformation may provide a dynamic model of hormone action and bone cell modulation in vitro.

Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis.

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg(2 + ) which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.

[Effects of heroin and purine nucleotide compensation on neutral alpha-glucosidase in rat epididymides].

OBJECTIVE: To study the changes of neutral alpha-glucoside activity in the epididymis of heroin-dependent and heroin-withdrawal rats, and to investigate the effects of intervention with purine nucleotide (AMP and GMP). METHODS: Eighty Wistar rats were randomly divided into 8 groups of equal number, control, nucleotide, heroin, heroin + nucleotide, 3 d withdrawal, 9 d withdrawal, 3 d nucleotide (nucleotide administrated for 3 days after heroin withdrawal) and 9 d nucleotide (nucleotide administrated for 9 days after heroin withdrawal). Neutral alpha-glucosidase activity in the epididymis was detected in each group of rats. RESULTS: Compared with the control group, neutral alpha-glucoside activity was markedly decreased in the heroin group (P < 0.05), and also in the 3 d and 9 d withdrawal groups, although with no significant differences (P > 0.05). CONCLUSION: Heroin reduces neutral alpha-glucoside activity in the epididymis of rats, and this effect may continue for some time after drug withdrawal, while purine nucleotide can keep neutral alpha-glucosidase activity in a relatively stable state.

Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5'-Nucleotidase (CD73) Inhibitors.

Cluster of differentiation 73 (CD73) converts adenosine 5'-monophosphate to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogues, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N3-methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N4-(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N4-benzoyl-cytidine (7f), N4-[ O-(4-benzyloxy)]-cytidine (9h), and N4-[ O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) ( Ki values 5-10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

Discovery and applications of nucleoside antibiotics beyond polyoxin.

Nucleoside antibiotics possess various biological activities such as antibacterial, antifungal, anticancer, and herbicidal activities. RIKEN scientists contributed to this area of research with two representative antifungal nucleoside antibiotics, blasticidin S and polyoxin. Blasticidin S was the first antibiotic exploited in agriculture worldwide. Meanwhile, the polyoxins discovered by Isono and Suzuki are still used globally as an agricultural antibiotic. In this review article, the research on nucleoside antibiotics mainly done by Isono and his collaborators is summarized from the discovery of polyoxin to subsequent investigations.

Uncoupling proteins: a role in protection against reactive oxygen species--or not?

A physiological function of the original uncoupling protein, UCP1, is well established: UCP1 is the molecular background for nonshivering thermogenesis. The functions of the "novel" UCPs, UCP2 and UCP3, are still not established. Recent discussions imply that all UCPs may play a role in protection against reactive oxygen species (ROS). Here we examine critically the evidence that UCP1, UCP2 and UCP3 are stimulated by ROS (superoxide) or ROS products (4-hydroxy-2-nonenal), and that the UCPs actually diminish oxidative damage. We conclude that, concerning UCP1, it is unlikely that it has such a role; concerning UCP2/UCP3, most evidence for physiologically significant roles in this respect is still circumstantial.