Methylthioadenosine deaminase in an alternative quorum sensing pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-α-d-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported [Guan, R., Ho, M. C., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 1247-1254], and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The k(cat)/K(m) value of 1.6 × 10(7) M(-1) s(-1) for MTA is the highest catalytic efficiency known for an MTA deaminase. 5'-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC(50) of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-α-d-ribose are involved in quorum sensing pathways; thus, PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8(R)-hydroxyl group in contact with a catalytic site Zn(2+), the 5'-methylthio group in a hydrophobic pocket, and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.

Structural basis for the growth factor activity of human adenosine deaminase ADA2.

Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions.

Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases.

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

Synthesis of 5'-methylthio coformycins: specific inhibitors for malarial adenosine deaminase.

Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2'-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5'-methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme (BtADA) can deaminate 5'-methylthioadenosine. 5'-Methylthiocoformycin and 5'-methylthio-2'-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA.

Relatively small increases in the steady-state levels of nucleobase deamination products in DNA from human TK6 cells exposed to toxic levels of nitric oxide.

Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 microM NO along with 186 microM O2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method, and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2'-deoxyuridine (dU) and 2'-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 microM NO and 186 microM O2 for 12 h (1260 microM x min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 h recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI, and dU, respectively, to reach the following levels: dX, 7 (+/- 1) per 10(6) nt; dI, 25 (+/- 2.1) per 10(6) nt; and dU, 40 (+/- 3.8) per 10(6) nt. dO was not detected above the limit of detection of 6 lesions per 10(7) nt in 50 microg of DNA. A 12 h exposure to 0.65 microM NO and 190 microM O2 (468 microM x min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a approximately 15% (+/- 3.6) reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA.

Dominant localization of adenosine deaminase in leptomeninges and involvement of the enzyme in sleep.

Adenosine is an endogenous hypnotic molecule. However, the mechanism by which the level of extracellular adenosine is regulated remains to be elucidated. We found by Northern hybridization and enzyme assay that ecto-5(')-nucleotidase and adenosine deaminase (ADA), major enzymes responsible for the production and degradation of adenosine, respectively, were localized most abundantly in the leptomeninges within the rat brain. Immunohistochemical study showed that ADA was dominantly localized in arachnoid barrier and trabecular cells of the leptomeninges. In vivo microdialysis demonstrated that externally applied adenosine was rapidly metabolized by ADA to inosine in the subarachnoid space. Perfusion of an ADA inhibitor, coformycin, increased the extracellular adenosine level in the subarachnoid space under the rostral basal forebrain. When coformycin was continuously infused into the subarachnoid space, non-rapid eye movement sleep was increased with prolonged duration of the sleep episode. These results demonstrate that the leptomeninges control the extracellular level of adenosine in the subarachnoid space by their high 5(')-nucleotidase and ADA activities and regulate non-rapid eye movement sleep.

Extracellular ATP and adenosine induce cell apoptosis of human hepatoma Li-7A cells via the A3 adenosine receptor.

1. Extracellular ATP is a potent signaling molecule that modulates a myriad of cellular functions through the activation of P2 purinergic receptors and is cytotoxic to a variety of cells at higher concentrations. The mechanism of ATP-elicited cytotoxicity is not fully understood. In this study, we investigated the effect of extracellular ATP on the human hepatoma Li-7A cells. 2. We observed a time- and dose-dependent growth inhibition of Li-7A cells by ATP, which is accompanied by an increase in the active form of caspase-3 as well as increased cleavage of its substrate, poly (ADP-ribose) polymerase. The cytotoxic effect of extracellular ATP was not mediated by the P2X7 receptor, since (1).the effect was not abolished by the P2X7 receptor antagonists oxidized ATP and KN-62, and (2).extracellular ADP, AMP, and adenosine were also cytotoxic. 3. We found that ATP and ADP were degraded to adenosine by Li-7A cells and that treatment of Li-7A cells by adenosine resulted in growth inhibition and caspase-3 activation, indicating that adenosine is the apoptotic agent. Using adenosine receptor agonists and antagonists, as well as inhibitors of adenosine transport and deamination, we showed that the cytotoxic effect of adenosine is specifically mediated by the A3 receptor even though transcripts of A1, A2A, A2B, and a splice variant of the P2X7 receptors were detected in Li-7A cells by RT-PCR. 4. Cytotoxicity caused by exogenous ATP and adenosine was completely abolished by the caspase-3 inhibitor Z-DEVD-FMK, demonstrating the central role of caspase-3 in apoptosis of Li-7A cells.

Enhancement of cellular adenosine triphosphate levels in PC12 cells by extracellular adenosine.

To elucidate the biological significance of extracellular adenine compounds, the effects of adenosine (Ado) on cellular levels of adenine compounds, especially adenosine triphosphate (ATP), in PC12 cells were studied. Ado and inosine but not adenosine 5'-monophosphate, adenosine 5'-diphosphate, ATP, guanosine, cytosine, thymidine, and uridine, significantly enhanced cellular ATP levels in PC12 cells in time- and dose-dependent manners. Various P1 receptor agonists of Ado did not enhance the ATP level. In addition, theophylline, an antagonist of P1 receptors, did not inhibit the Ado-evoked ATP enhancement. These results suggest that the Ado receptor is not involved in the augmentation of the cellular ATP level induced by Ado in PC12 cells. The ATP-enhancing effect of Ado was potentiated by dipyridamole, an inhibitor of Ado uptake, or coformycin, an inhibitor of Ado deaminase. The effect of Ado on the ATP level was also observed when PC12 cells were incubated in glucose-free medium. Together these results suggest that enhancement of cellular ATP levels in PC12 cells by extracellular Ado might be acceleration of ATP synthesis through the Ado salvage system using hypoxanthine-guanine phosphoribosyltransferase rather than Ado kinase since 5'-iodotubercidin, an inhibitor of Ado kinase, had no effect on the enhancement elicited by Ado.

Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice.

We have used an enzyme-based, twin-barrelled sensor to measure adenosine release during hypoxia in the CA1 region of rat hippocampal slices in conjunction with simultaneous extracellular field recordings of excitatory synaptic transmission. When loaded with a combination of adenosine deaminase, nucleoside phosphorylase and xanthine oxidase, the sensor responded linearly to exogenous adenosine over the concentration range 10 nM to 20 microM. Without enzymes, the sensor when placed on the surface of hippocampal slices recorded a very small net signal during hypoxia of 40 +/- 43 pA (mean +/- s.e.m.; n = 7). Only when one barrel was loaded with the complete sequence of enzymes and the other with the last two in the cascade did the sensor record a large net difference signal during hypoxia (1226 +/- 423 pA; n = 7). This signal increased progressively during the hypoxic episode, scaled with the hypoxic depression of the simultaneously recorded field excitatory postsynaptic potential and was greatly reduced (67 +/- 6.5 %; n = 9) by coformycin (0.5-2 microM), a selective inhibitor of adenosine deaminase, the first enzyme in the enzymic cascade within the sensor. For 5 min hypoxic episodes, the sensor recorded a peak concentration of adenosine of 5.6 +/- 1.2 microM (n = 16) with an IC(50) for the depression of transmission of approximately 3 microM. In slices pre-incubated for 3-6 h in nominally Ca(2+)-free artificial cerebrospinal fluid, 5 min of hypoxia resulted in an approximately 9-fold greater release of adenosine (48.9 +/- 17.7 microM; n = 6). High extracellular Ca(2+) (4 mM) both reduced the adenosine signal recorded by the sensor during hypoxia (3.5 +/- 0.6 microM; n = 4) and delayed the hypoxic depression of excitatory synaptic transmission.

Purine analogues kill resting lymphocytes by p53-dependent and -independent mechanisms.

To resolve the controversy concerning the role of p53 in the killing of resting lymphocytes by purine nucleoside analogues, we examined the cytotoxic effects of chlorodeoxyadenosine, fludarabine and deoxycoformycin (plus deoxyadenosine) on unstimulated spleen cells from p53-knockout versus wild-type mice. p53-knockout cells were more resistant to all three nucleosides than were wild-type cells. However, substantial killing still occurred in the absence of p53, indicating that purine analogues can kill resting lymphocytes by both p53-dependent and -independent mechanisms. We suggest that these results are relevant to chronic lymphoid malignancies, and that characterization of the p53-independent component of nucleoside action may indicate potential ways of overcoming therapeutic resistance.

Adenine nucleotide synthesis in human erythrocytes depends on the mode of supplementation of cell suspension with adenosine.

In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.

5'-Nucleotidase as a marker of both general and local inflammation in rheumatoid arthritis patients.

OBJECTIVES: To evaluate measurements of serum and synovial fluid 5'-nucleotidase (5'N) activity as a marker of general and local inflammation in arthritis, and to resolve a contradiction in the literature as to whether or not the activity of 5'N in the synovial fluids of rheumatoid arthritis (RA) patients is raised in comparison with that in the synovial fluids of other arthritis patients. METHODS: Assays for 5'N were carried out in the presence of inhibitors of other phosphatases, AMP deaminase and of 5'N itself. RESULTS: The 5'N activity in the synovial fluid of RA patients was both significantly higher (mean 1.7-fold) and had a greater variance than that in the synovial fluids of other arthritis patients, and the contradiction in the literature was resolved. There was a strong correlation between the 5'N activity in the sera of RA patients and their erythrocyte sedimentation rate. There was no significant correlation between the 5'N in the serum and synovial fluid for the RA patients, in marked contrast to the strong correlation between the two 5'N activities shown by the osteoarthritis patients. The 5'N activity was greater in the synovial fluid than in the serum for virtually all the patients, showing that it was being made locally. CONCLUSIONS: The 5'N activity in the serum (which came mostly from the liver) could be used as a marker of general inflammation, whereas the 5'N in the synovial fluid was mostly produced locally, and could be used as a marker of joint inflammation, particularly for the RA patients.

Delayed production of adenosine underlies temporal modulation of swimming in frog embryo.

1. To investigate the dynamics of adenosine production in the spinal cord during motor activity, and its possible contribution to the temporal modulation of motor patterns, a sensor sensitive to adenosine at concentrations as low as 10 nM was devised. 2. When pressed against the outside of the spinal cord, the sensor detected slow changes in the levels of adenosine during fictive swimming that ranged from 10 to 650 nM. In four embryos where particularly large signals were recorded due to favourable probe placement, the adenosine levels continued to rise for up to a minute following cessation of activity before slowly returning to baseline. In the remaining thirteen embryos, levels of adenosine started to return slowly to baseline almost immediately after activity had stopped. 3. Inhibitors of adenosine uptake increased the magnitude of the signal recorded and slowed the recovery following cessation of activity. 4. A realistic computational model of the spinal circuitry was combined with models of extracellular breakdown of ATP to adenosine. ATP and adenosine inhibited, as in the real embryo, the voltage-gated K+ and Ca2+ currents, respectively. The model reproduced the temporal run-down of motor activity seen in the real embryo suggesting that synaptic release of ATP together with its extracellular breakdown to adenosine is sufficient to exert time-dependent control over motor pattern generation. 5. The computational analysis also suggested that the delay in the rise of adenosine levels is likely to result from feed-forward inhibition of the 5'-ectonucleotidase in the spinal cord. This inhibition is a key determinant of the rate of run-down.

Adenosine, inosine, and guanosine protect glial cells during glucose deprivation and mitochondrial inhibition: correlation between protection and ATP preservation.

The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 microM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 microM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 microM BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.

Evidence for a low temperature transition state binding preference in bovine adenosine deaminase.

Arrhenius plots of the interactions of bovine adenosine deaminase (ADA) and of coformycin-inhibited ADA with adenosine are non-linear and reveal that coformycin significantly increases the activation energy for reaction only at temperatures well below the normal operating temperature of the enzyme (38.3 degrees C). This apparent enhanced affinity of the enzyme for the transition state analog at low temperature is confirmed from determinations of coformycin binding at 38.3 degrees C (KI = 5.3 x 10(-11) M) and at 21 degrees C (KI = 1.1 x 10(-11) M). It is suggested that these data are inconsistent with a model for general enzyme catalysis that requires an initial transition state complementary active site. Instead, it is suggested that an initial active site transition state complementarity is undesirable and the tendency of the enzyme to exist in this conformer at low temperatures is responsible for its inefficient interaction with adenosine substrate.

The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine.

The purine nucleoside adenosine (9-beta-D-ribofuranosyladenine) inhibits a number of lymphocyte functions in vitro, including the ability of activated T lymphocytes and natural killer cells to adhere to and kill tumor targets. Solid tumors, such as adenocarcinomas of the lung and colon, are frequently hypoxic and are, therefore, likely to exhibit increased adenine nucleotide breakdown through the 5'-nucleotidase pathway, yielding adenosine. We examined whether the concentration of adenosine in the extracellular fluid of such tumors is adequate to cause immunosuppression. Murine tumors grown in syngeneic hosts or human tumors grown in immunodeficient nu/nu mice were subjected to microdialysis, and adenosine levels in the microdialysate were measured by high-performance liquid chromatography. Treatment of the tumor microdialysates with adenosine deaminase eliminated the adenosine peak. Recovery of adenosine ranged from 15 to 29%, depending on the microdialysis probe, and concentrations of adenosine in tumors ranged from 0.2 to 2.4 microM with a mean of 0.5 microM. In contrast, the adenosine concentration measured s.c. at the same location was 30 +/- 5 nM (mean +/- SE). Inclusion of the adenosine deaminase inhibitor coformycin (10 microM) and the adenosine kinase inhibitor 5'-iodotubercidin (0.1 microM) in the microdialysis perfusion buffer increased extracellular adenosine concentration in tumors to as high as 13 microM. These data show that extracellular adenosine levels in solid tumors are sufficient to suppress the local antitumor immune response and that interference with pathways of adenosine metabolism causes marked increases in tumor extracellular adenosine concentration.

Adenosine-5'-phosphate deaminase. A novel herbicide target.

The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool.

Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons.

Drug-selected intrachromosomal gene amplification by breakage-fusion-bridge (BFB) cycles is well documented in mammalian cells, but factors governing this mechanism are not clear. Here, we show that only some clastogenic drugs induce drug resistance through intrachromosomal amplification. We strictly correlate triggering of BFB cycles to induction of fragile site expression. We demonstrate a dual role for fragile sites in intrachromosomal amplification: a site telomeric to the selected gene is involved in initiation, while a centromeric site defines the size and organization of early amplified units. The positions of fragile sites relative to boundaries of amplicons found in human cancers support the hypothesis that fragile sites play a key role in the amplification of at least some oncogenes during tumor progression.

Purine nucleotide cycle in rat renal cortex and medulla under conditions that mimic normal and low oxygen supply.

The distinctive feature of the renal function and metabolism implicate a possibility of excessive ATP degradation during insufficient oxygen supply. Protection of the purine ring against degradation is one among other functions of the purine nucleotide cycle (PNC). The purpose of this study was to estimate the activity of PNC in cytosol of rat renal cortex and medulla under conditions that mimic normal and low oxygen supply in vivo. In normoxic-like condition the rate of AMP deamination was 1.7 and 2.0 nmol/mg protein/min in the cytosol of cortex and medulla, respectively. Under this condition, the rate of IMP reamination was similar to that of AMP deamination. In a hypoxia-like condition the rate of AMP deamination increased by 41% in cytosol from both parts of the kidney, while the rate of IMP reamination remained unchanged in the cytosol of medulla and decreased by 46% in the cortex cytosol. Distribution of the other enzymes of the PNC, that is, adenylosuccinate synthetase and adenylosuccinate lyase, in the cytosol of cortex and medulla correlated with that observed for AMP deamination and IMP reamination potentials. At 150 microM IMP, the activity of adenylosuccinate synthetase in the cortex and medulla was 0.34 and 1.24 nmol/mg protein/min, respectively. Activity of the adenylosuccinate lyase was severalfold greater than the respective activity of the adenylosuccinate synthetase. These results show that the efficiency of PNC is about twice as high in the medulla cytosol as in the cortex cytosol, and that the activity of PNC in kidney is mainly limited by the activity of adenylosuccinate synthetase and supply of AMP.