Pixel-based absolute topography test for three flats.

We demonstrate a method of performing the absolute three-flat test by using reflection symmetries of the surfaces and an algorithm for generating the rotation of arrays of pixel data. Most of the operations involve left/right and top/bottom flips of data arrays, operations that are very fast on most frame grabbers and are available on most commercial phase-measuring interferometers. We demonstrate the method with simulated data as well as with actual data from 150-mm-diameter surfaces that are flat to less than 25 nm peak to valley.

Purine nucleoside phosphorylase. 3. Reversal of purine base specificity by site-directed mutagenesis.

Human purine nucleoside phosphorylase (PNP) is highly specific for 6-oxopurine nucleosides with a catalytic efficiency (kcat/KM) for inosine 350000-fold greater than for adenosine. Crystallographic studies identified Asn243 and Glu201 as the residues largely responsible for the substrate specificity. Results from mutagenesis studies demonstrated that the side chains for both residues were also essential for efficient catalysis [Erion, M. D., et al. (1997a) Biochemistry 36, 11725-11734]. Additional mechanistic studies predicted that Asn243 participated in catalysis by stabilizing the transition state structure through hydrogen bond donation to N7 of the purine base [Erion, M. D., et al. (1997b) Biochemistry 36, 11735-11748]. In an effort to alter the substrate specificity of human PNP, mutants of Asn243 and Glu201 were designed to reverse hydrogen bond donor and acceptor interactions with the purine base. Replacement of Asn243 with Asp, but not with other amino acids, led to a 5000-fold increase in kcat for adenosine and a 4300-fold increase in overall catalytic efficiency. Furthermore, the Asn243Asp mutant showed a 2.4-fold preference for adenosine relative to inosine and a 800000-fold change in substrate specificity (kcat/KM) relative to wild-type PNP. The double mutant, Asn243Asp::Glu201Gln, exhibited a 190-fold increase in catalytic efficiency with adenosine relative to wild-type PNP, a 480-fold preference for adenosine relative to inosine, and a 1.7 x 10(8)-fold change in preference for adenosine over inosine relative to wild-type PNP. The Asn243Asp mutant was also shown to synthesize 2,6-diaminopurine riboside with a catalytic efficiency (1.4 x 10(6) M-1 s-1) on the same order of magnitude as wild-type PNP with its natural substrates hypoxanthine and guanine. The Asn243Asp mutants represent examples in which protein engineering significantly altered substrate specificity while maintaining high catalytic efficiency.

Test of a slow off-axis parabola at its center of curvature.

We describe the interferometric testing of a slow (ƒ/16 at the center of curvature) off-axis parabola, intended for use in an x-ray spectrometer, that uses a spherical wave front matched to the mean radius of the asphere. We find the figure error in the off-axis mirror by removing the theoretical difference between the off-axis segment and the spherical reference from the measured wave-front error. This center of curvature test is easy to perform because the spherical reference wave front has no axis and thus alignment is trivial. We confirm that the test results are the same as the double-pass null test for a parabola that uses a plane autocollimating mirror. We also determine that the off-axis section apparently warped as the result of being cut from the symmetric parent part.

Visualization of surface figure by the use of Zernike polynomials.

Commercial software in modern interferometers used in optical testing frequently fit the wave-front or surface-figure error to Zernike polynomials; typically 37 coefficients are provided. We provide visual representations of these data in a form that may help optical fabricators decide how to improve their process or how to optimize system assembly.

Platelet-activating factor (PAF)-induced platelet aggregation. Modulation by plasma adenosine and methylxanthines.

This study examined the role of plasma adenosine in the modulation of platelet-activating factor (PAF) activity on platelet aggregation and serotonin (5-HT) release in human platelet-rich plasma (PRP). In addition, the effects of methylxanthines (e.g. theophylline and caffeine) were studied on PAF-induced platelet aggregation in PRP isolated from blood samples from healthy subjects. Also, PAF-induced platelet aggregation was examined in PRP samples of patients receiving theophylline treatment. These studies demonstrate that plasma adenosine levels (0.1 to 0.3 microM) play a key role in negative modulation of PAF activity on platelet aggregation and 5-HT release. After depletion of plasma adenosine, the platelet-aggregating activity of PAF was increased greatly (> 10-fold). PAF at concentrations of 0.1 to 12 microM caused no 5-HT release in PRP containing normal amounts of adenosine (blood collected in the presence of 2'-deoxycoformycin and dilazep), whereas PAF at 0.1 microM caused 5-HT release (45%) in adenosine-depleted PRP, demonstrating that plasma adenosine is much more inhibitory of 5-HT release than platelet aggregation. The adenosine antagonists theophylline (50 microM), caffeine (50 microM) and a xanthine derivative, 3,7-dimethyl-l-propargylxanthine (DMPX, 10 microM) (a more specific adenosine A2 receptor antagonist), potentiated PAF activity on platelet aggregation in PRP samples containing adenosine. Also, patients receiving theophylline treatments showed significantly greater platelet aggregation induced by PAF in their PRP samples. PAF induced a rapid increase (80% in 15 sec) in intracellular Ca2+ mobilization, which was strongly inhibited by adenosine (IC50, 0.3 microM). Our studies suggest that agents that can increase plasma adenosine levels (e.g. inhibitors of adenosine uptake and adenosine metabolism) or methylxanthines may be useful in altering (inhibiting or enhancing, respectively) PAF actions on platelets and other tissues.

Plasma adenosine deaminase2: a marker for human immunodeficiency virus infection.

Plasma concentrations of the two isoenzymes of adenosine deaminase (ADA, E.C. 3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), were measured in a cohort of ambulatory patients infected with the human immunodeficiency virus (HIV) and controls. A sensitive isoenzyme-specific radioisotopic assay system was developed for these studies. Among 22 HIV-infected patients, plasma ADA2 was significantly elevated as compared with 16 control subjects (p less than 0.01) and 6 uninfected subjects having a risk factor for HIV infection (p less than 0.01). Plasma ADA2 was not associated with the stage of disease as defined by clinical status (p greater than 0.05) or helper (CD4) lymphocyte count (p greater than 0.05). Available evidence suggests that elevated plasma ADA2 could be a useful surrogate marker for HIV infection that occurs early in the disease process.

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme.

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.

Inhibition of nucleoside transport by nitrobenzylthioformycin analogs.

The formycin analogs of nitrobenzylthioinosine and nitrobenzylthioguanosine were synthesized and evaluated as nucleoside transport inhibitors. These analogs have a potential therapeutic advantage over their parent compounds in that their C-nucleosidic linkages prevent them from being degraded to the immunosuppressive agents, 6-mercaptopurine and 6-thioguanine. 7-[(4-Nitrobenzyl)-thio]-3-(beta-D-ribofuranosyl)pyrazolo[4,3- d]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(beta-D- ribofuranosyl)pyrazolo[4,3-d]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC50 value for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [3H]NBTF yielded a Kd of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with Kd values of 8.1 and 0.81 nM respectively. The octanol/water partition coefficients of nitrobenzylthioinosine, NBTF and NBTGF were 3.5, 3.2, and 2.8 respectively. NBTF could be expected to be equipotent with nitrobenzylthioinosine in whole blood where inhibitor concentrations of 10(-7) to 10(-6) M are required in order to saturate erythrocytic binding sites; hence, it may exhibit the advantages inherent in a C-nucleoside.

Three-dimensional structure of human erythrocytic purine nucleoside phosphorylase at 3.2 A resolution.

The three-dimensional structure of human erythrocytic purine nucleoside phosphorylase has been determined at 3.2 A resolution using x-ray diffraction data. Intensity data were measured using radiation from the Synchrotron Radiation Source, Daresbury, England, and oscillation film techniques. Phases were determined by using multiple isomorphous replacement methods with four heavy-atom derivatives and were improved using solvent flattening techniques. Purine nucleoside phosphorylase exists in the crystal as a trimer in which subunits are related by a crystallographic 3-fold axis. Each subunit contains an eight-stranded mixed beta-sheet and a five-stranded mixed beta-sheet which join to form a distorted beta-barrel structure. This core beta-structure is flanked by seven alpha-helices in a manner that generates a novel folding pattern. The active site, which was characterized from binding of the substrate analogs 8-iodoguanine and 5'-iodoformycin B, is located near the subunit-subunit boundary within the trimer and involves seven different segments from one subunit and an additional short segment from an adjacent subunit. In the crystal, the phosphate-binding site is probably occupied by a sulfate ion. The specificity of purine nucleoside phosphorylase for guanine, hypoxanthine, and their analogs can be explained on the basis of the arrangement of hydrogen bond donors and acceptors in the active site.

Mirror deflection on multiple axial supports.

A theoretical model based on thin plate theory is developed to calculate mirror deflection on multiple axial supports. Thickness variation due to mirror curvature, sandwich structure, and shear effects is included in this model. Comparison with the finite element analysis shows almost identical results for a three-point support. Surface deflections due to more complicated support patterns (forty-eight- and eighty-four-point support) are also included. A tolerancing model of support forces is developed and applied to the case of eight-four-point support.

Clinical, pharmacologic, and immunologic effects of 2'-deoxycoformycin.

Clinical, pharmacologic, and immunologic effects of 2'-deoxycoformycin (dCF) were evaluated in 15 patients with advanced malignancies. Toxicity was less severe with a low dose (4 mg/m2) of dCF, but this dose still resulted in suppression of cellular adenosine deaminase activity, skin test reactivity, and lymphocyte responses to mitogens. Improvement in cutaneous T cell lymphoma plaques was seen after dCF. Further investigations of antitumor efficacy with the use of this low dosage schedule should continue in patients with hematologic neoplasms, and additional preliminary studies of the combination of an adenosine deaminase inhibitor with an adenosine analog should also be considered.

Adenosine metabolism in human whole blood. Effects of nucleoside transport inhibitors and phosphate concentration.

Adenosine (Ado, 10 microM) was metabolized in whole blood within 1 min, primarily to hypoxanthine and ATP. The concentration of Ado, the activities of adenosine deaminase (ADA) and Ado kinase, the Km values for Ado with ADA and Ado kinase, and the substrate inhibition of Ado kinase are factors that govern the Ado metabolism between deamination and phosphorylation. If ADA activity was blocked by 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of ADA, most of the Ado (96%) was incorporated into adenine nucleotides, whereas if Ado kinase activity was blocked with 5-iodotubercidin (10 microM), Ado was mainly (95%) metabolized into hypoxanthine. A high phosphate concentration (25 mM) caused marked increases in the formation of IMP. The nucleoside transport inhibitors dilazep (1 microM), dipyridamole (10 microM) and nitrobenzylthioinosine (NBMPR, 1 microM) strongly blocked cellular Ado metabolism. In the presence of nucleoside transport inhibitors, Ado which slowly enters the cell was metabolized principally by Ado kinase rather than ADA. Dilazep, NBMPR and dipyridamole were more effective in blocking Ado uptake and metabolism by erythrocytes suspended in a protein-free medium than by cells suspended in plasma.

Substrate specificities of 5'-deoxy-5'-methylthioadenosine phosphorylase from Trypanosoma brucei brucei and mammalian cells.

The separation by chromatofocusing of two distinct purine nucleoside cleaving activities from crude extracts of Trypanosoma brucei brucei is described. One catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MeSAdo) and adenosine (Ado) and was designated an MeSAdo/Ado phosphorylase, while the other catalyzes the hydrolysis of adenosine, inosine, and guanosine but not MeSAdo. The substrate specificity of trypanosomal MeSAdo/Ado phosphorylase differed from that of a mammalian MeSAdo phosphorylase (derived from murine Sarcoma 180 cells) in that it was able to phosphorolyze 2'-deoxyadenosine, 3'-deoxyadenosine and 2',3'-dideoxyadenosine. In addition, the trypanosomal phosphorylase was able to utilize the nucleoside analog, 6-methylpurine 2'-deoxyribonucleoside, as an alternative substrate, whereas the mammalian enzyme could not. Because of these differences, cytotoxic analogs of MeSAdo may be designed that are selectively activated by the trypanosomal MeSAdo/Ado phosphorylase.

Role of plasma adenosine in the antiplatelet action of HL 725, a potent inhibitor of cAMP phosphodiesterase: species differences.

The potent inhibitor of platelet cAMP phosphodiesterase (PDE) HL 725 (9,10-Dimethoxy-2-mesitylimino-3-methyl-3, 4,6,7-tetrahydro-2H-pyrimido(6,1-A)-isoquinoline-4-one-hydrochloride), was examined for its effects on human and rat platelet aggregation. Strong inhibitory effects are seen on collagen-induced platelet aggregation both in rat platelet-rich plasma (PRP) (IC50, 54 +/- 12 nM) and whole blood (IC50, 57 +/- 25 nM). Compared to the effects on rat platelets, HL 725 is about two-fold less inhibitory in human PRP (IC50, 94 +/- 29 nM) and whole blood (IC50, 126 +/- 50 nM). The inhibitory action of HL 725 can be reversed by washing and resuspension of the platelets, suggesting that HL 725 does not bind tightly to cAMP PDE. If human or rat PRP is pretreated with adenosine deaminase, an enzyme that degrades adenosine or 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, the inhibitory effect of HL 725 is reversed. Similar blockade of the inhibitory actions of several other inhibitors of cAMP PDE such as RA 233, RX-RA 69 (analogs of dipyridamole) and oxagrelate is seen by adenosine deaminase pretreatment. The nucleoside transport inhibitors, dilazep and dipyridamole which are non-inhibitory alone to platelet aggregation, strongly potentiate (about 10-fold) the inhibitory action of HL 725 on collagen-induced platelet aggregation in human whole blood. However, if the whole blood is pretreated with adenosine deaminase, no inhibitory effect of dipyridamole plus HL 725 is seen on platelet aggregation. These studies demonstrate that plasma adenosine plays a crucial role in the antiaggregatory actions of HL 725 and several other inhibitors of cAMP PDE both in human and rat blood.

5'-deoxy-5'-methylthioadenosine phosphorylase--IV. Biological activity of 2-fluoroadenine-substituted 5'-deoxy-5'-methylthioadenosine analogs.

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methylthioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5'-deoxy-5'-iodo-2-fluoroadenosine (5'-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5'-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds (IC50 values in the 10(-8) M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/aprt1 cells) did not form analog nucleotides and were relatively insensitive to these compounds (IC50 values in the 10(-5) M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of S-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.

Inhibition of human purine nucleoside phosphorylase by acyclic nucleosides and nucleotides.

In an effort to develop more potent inhibitors of human purine nucleoside phosphorylase (PNP) as immunosuppressive and cancer chemotherapeutic agents, the affinity of the erythrocytic enzyme for 30 acyclic nucleosides, nucleotides and related compounds was determined. Among the acyclonucleosides, 2'-nordeoxyguanosine [2'NDG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine] had a 3-fold greater affinity than acyclovir, and 8-amino-2'NDG was the best inhibitor with Ki = 2.6 X 10(-7) M. The ether moiety of the acyclovir and 2'NDG side-chains was not important for binding. Phosphorylated 2'NDG analogs appeared to act as multisubstrate analogs with optimal binding at low (1 mM) phosphate concentration. The 2'NDG mono- and triphosphates had higher affinities than those reported for the phosphorylated acyclovir derivatives but the diphosphate had a similar Ki value of 9 X 10(-9) M. Poor affinity, independent of phosphate concentration, was found for 9-(2-phosphonoethyl)guanine. The 3'-phosphate derivative of 8-(3-hydroxypropyl)-9-methylguanine inhibited with a Ki = 2 X 10(-5) M in 1 mM phosphate. The chemical syntheses of new analogs are described.

Murine tumor-induced platelet aggregation and coagulation: mechanisms, inhibitors, and species differences.

This study examined the platelet-aggregating and procoagulant activities of two hematogenously disseminating tumors, a mouse lymphoblastic leukemia (L5178Y) and a mouse renal adenocarcinoma (RAG). Tumor-induced human platelet aggregation was inhibited by addition of the following agents to platelet-rich plasma (PRP): a calcium channel blocker (verapamil), a chelator of divalent cations (EDTA), stimulators of adenylate cyclase (2-fluoroadenosine and forskolin), and inhibitors of cAMP phosphodiesterase (oxagrelate and papaverine). The platelet-aggregating activities of both cell lines were completely blocked by treatment of the cells with heat, sonication, phospholipase A2, and Triton X-100. These data suggest that L5178Y and RAG cell-induced human platelet aggregation are dependent on a heat-labile phospholipid component of the tumor cell membrane. L5178Y cells had greater platelet-aggregating activity in human plasma than in rat or mouse plasma, whereas RAG cells had greater procoagulant activity in rat or mouse plasma than in human plasma. The procoagulant activity of RAG cells in rat and mouse plasma was demonstrated by three lines of evidence: RAG cells induced heparinized PRP to clot; the thrombin inhibitor DAPA lengthened of the clotting time and the lag time before aggregation; and RAG cells shortened of the recalcification time of the plasma. The above data indicate that RAG cell-induced murine platelet aggregation and coagulation is dependent on thrombin generation.

Design of purine nucleoside phosphorylase inhibitors.

Purine nucleoside phosphorylase inhibitors hold promise as specific immunosuppressive, anti-T cell leukemic, and antiuricopoietic agents. The best inhibitors available that are biologically active have Ki values from 10(-6) to 10(-7) M and fall into two categories: noncleavable nucleosides preferably iodinated at the C-5' position and C-8-substituted guanine or acycloguanosines. More potent inhibition is shown by phosphorylated acyclonucleosides that function as multisubstrate analogs, but these compounds are excluded from cells. The X-ray analysis of the human erythrocytic enzyme is beginning to reveal the nature of the active site and to explain the structure-activity relationships that have been established with analog substrates and inhibitors.

Potentiation of the antiplatelet action of adenosine in whole blood by dipyridamole or dilazep and the cAMP phosphodiesterase inhibitor, RA 233.

Adenosine (Ado, 10-50 microM), a potent inhibitor of ADP-induced human platelet aggregation in platelet-rich plasma (PRP), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) which are resistant to deamination (2-fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibit aggregation in whole blood with IC50 values of 12 microM, 2.3 microM and 0.26 microM, respectively. The inhibitory effect of NECA (200 nM) is potentiated by the platelet cAMP phosphodiesterase (PDE) inhibitor RA 233 (5 microM). Inhibition of the erythrocytic nucleoside transport system by dilazep (1 microM) or dipyridamole (10 microM), or blockade of Ado metabolism by 2'-deoxycoformycin (5 microM) plus 5-iodotubercidin (10 microM), evokes the antiaggregatory action of Ado in whole blood (IC50 congruent to 2 microM). RA 233 (5 microM) potentiates Ado-mediated inhibition about 10-fold when nucleoside transport or Ado metabolism is blocked. Ado (10 microM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleoside transport is inhibited by dilazep or dipyridamole, or when Ado metabolism is blocked by 2'-deoxycoformycin and 5-iodotubercidin, 50-60% of the Ado remains in the plasma after 5 min. These results show that the failure of Ado to inhibit platelet aggregation in whole blood results from its rapid uptake and metabolism by erythrocytes. More importantly, these data emphasize the key role of nucleoside transport inhibition in the antiplatelet actions of dipyridamole and dilazep. In addition, superior therapeutic results may be obtained from the combination of blockade of nucleoside transport system with inhibition of platelet cAMP PDE.

Changes in nucleoside transport of HL-60 human promyelocytic cells during N,N-dimethylformamide induced differentiation.

The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.