Effects of intravenous exenatide in type 2 diabetic patients with congestive heart failure: a double-blind, randomised controlled clinical trial of efficacy and safety.

AIMS/HYPOTHESIS: The aim of this study was to determine whether exenatide improves haemodynamic function in patients with type 2 diabetes with congestive heart failure (CHF). METHODS: The main eligibility criteria for inclusion were: male/female (18-80 years) with type 2 diabetes and CHF (ejection fraction ≤ 35%, and New York Heart Association functional class III or IV). Out of 237 patients screened, 20 male type 2 diabetic patients participated in this crossover trial design and were allocated (sequentially numbered) to i.v. infusions during two consecutive days with (1) exenatide (0.12 pmol/kg/min); and (2) placebo for 6 h followed by a washout period for 18 h, at Stockholm South Hospital, Sweden. Patients and researchers were blinded to the assignment. Cardiac haemodynamic variables were determined by right heart catheterisation. The primary endpoint was defined as an increase in cardiac index (CI) or a decrease in pulmonary capillary wedge pressure (PCWP) of ≥ 20%. Secondary endpoints were tolerability and safety of exenatide infusion. RESULTS: CI increased at 3 and 6 h by 0.4 ± 0.1 (23%) and 0.33 ± 0.1 (17%) l min(-1) m(-2), during exenatide infusion vs -0.02 ± 0.1 (-1%) and -0.08 ± 0.1 (-5%) l min(-1) m(-2) during placebo (p = 0.003); and heart rate (HR) increased at 1, 3 and 6 h by 8 ± 3 (11%), 15 ± 4 (21%) and 21 ± 5 (29%) beats per min (bpm), during exenatide infusion vs -1 ± 2 (-2%), 1 ± 1 (2%) and 6 ± 2 (8%) bpm, during placebo (p = 0.006); and PCWP decreased at 1, 3 and 6 h by -1.3 ± 0.8 (-8%), -1.2 ± 1 (-8%) and -2.2 ± 0.9 (-15%) mmHg, during exenatide infusion vs 0.3 ± 0.5 (2%), 1 ± 0.6 (6%) and 1.4 ± 0.7 (8%) mmHg, during placebo (p = 0.001). No serious adverse event was observed. Adverse events were reported in nine patients (six, nausea; two, increased HR; one, increased systolic blood pressure). CONCLUSIONS/INTERPRETATION: Infusion of exenatide in male type 2 diabetic patients with CHF increased the CI as a result of chronotropy, with concomitant favourable effects on PCWP and reasonable tolerability of the drug. The clinical implications of using exenatide in patients with CHF are still not clear and further studies are warranted. TRIAL REGISTRATION: www.isrctn.org/ISRCTN47533126

Specific cytotoxicity of arabinosylguanine toward cultured T lymphoblasts.

Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe T cell immunodeficiency. To understand further and exploit this T cell lymphospecificity, we have compared the cytotoxicities and metabolism of deoxyguanosine, the cytotoxic substrate of PNP and of arabinosylguanine, a deoxyguanosine analogue that is resistant to PNP cleavage, in T cell (8402) and B cell (8392) lines in continuous culture established from the same patient. In comparative growth rate experiments the T cells were 2.3-fold and 400-fold more sensitive to growth inhibition by deoxyguanosine and arabinosylguanine, respectively, than were the B cells. Only the T cells, but not the B cells, could phosphorylate in situ deoxyguanosine or arabinosylguanine to the corresponding triphosphate. Both the phosphorylation and cytotoxicity of arabinosylguanine in the T cell line could be prevented by deoxycytidine, which suggests that deoxycytidine-deoxyguanosine kinase initiated the intracellular metabolism and cytotoxicity of this nucleoside analogue. The sensitivity and selectivity of arabinosylguanine toward the T lymphoblastoid cells suggests a rational approach to the design of chemotherapeutic agents that are directed toward T cell malignancies and other T cell disorders.

Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.

A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage.

Expression of the high-affinity purine nucleobase transporter in mutant mouse S49 cells does not require a functional wild-type nucleoside-nucleobase transporter.

A novel type of somatic mutation that causes the expression of a high-affinity purine base permease (B. Aronow, D. Toll, J. Patrick, P. Hollingsworth, K. McCartan, and B. Ullmann, Mol. Cell Biol. 6:2957-2962, 1986) has been inserted into nucleoside transport-deficient S49 cells. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possessed an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possessed an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells could transport nucleosides, suggesting that the expression of the high-affinity base transporter did not reverse the mutation in the nucleoside transport system. The transport of purine bases by both AE1HADPAB2 and AE1HADPAB5 cells was much less sensitive than that by wild-type cells to inhibition by dipyridamole, 4-nitrobenzylthionosine, and N-ethylmaleimide, potent inhibitors of nucleoside and nucleobase transport in wild-type S49 cells. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild-type cells indicated that the expression of the high-affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high-affinity purine base transporter of mutant cells and the nucleoside transport function of wild-type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild-type cells.

Incomplete nucleoside transport deficiency with increased hypoxanthine transport capability in mutant T-lymphoblastoid cells.

From a mutagenized population of wild-type mouse (S49) T-lymphoma cells, a clone, 80-5D2, was isolated in a single step by virtue of its ability to survive in 80 nM 5-fluorouridine. Unlike previously isolated nucleoside transport-deficient cell lines (A. Cohen, B. Ullman, and D. W. Martin, Jr., J. Biol. Chem. 254:112-116, 1979), 80-5D2 cells were only slightly less sensitive to growth inhibition by a variety of cytotoxic nucleosides and were capable of proliferating in hypoxanthine-amethopterin-thymidine-containing medium. The molecular basis for the phenotype of 80-5D2 cells was incomplete deficiency in the ability of the mutant cells to translocate nucleosides across the plasma membrane. Interestingly, mutant cells were more capable than wild-type cells of transporting the nucleobase hypoxanthine. Residual transport of adenosine into 80-5D2 cells was just as sensitive to inhibition by nucleosides and more sensitive to inhibition by hypoxanthine than that in wild-type cells, indicating that the phenomena of ligand binding and translocation can be uncoupled genetically. The 80-5D2 cells lacked cell surface binding sites for the potent inhibitor of nucleoside transport p-nitrobenzylthioinosine (NBMPR) and, consequently, were largely resistant to the physiological effects of NBMPR. However, the altered transporter retained its sensitivity to dipyridamole, another inhibitor of nucleoside transport. The biochemical phenotype of the 80-5D2 cell line supports the hypothesis that the determinants that comprise the nucleoside carrier site, the hypoxanthine carrier site, the NBMPR binding site, and the dipyridamole binding site of the nucleoside transport function of mouse S49 cells are genetically distinguishable.

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

Expression of a novel high-affinity purine nucleobase transport function in mutant mammalian T lymphoblasts.

The single nucleoside transport function of mouse S49 lymphoblasts also transports purine bases (B. Aronow and B. Ullman, J. Biol. Chem. 261:2014-2019, 1986). This transport of purine bases by S49 cells is sensitive to inhibition by dipyridamole (DPA) and 4-nitrobenzylthioinosine, two potent inhibitors of nucleoside transport. Therefore, wild-type S49 cells cannot salvage low hypoxanthine concentrations in the presence of 10 microM DPA and 11 microM azaserine; the latter is a potent inhibitor of purine biosynthesis. Among a mutagenized wild-type population, a cell line, JPA2, was isolated which could proliferate in 50 microM hypoxanthine-11 microM azaserine-10 microM DPA. The basis for the survival of JPA2 cells under these selective conditions was expression of a unique, high-affinity purine nucleobase transport function not present in wild-type cells. JPA2 cells could transport 5 microM concentrations of hypoxanthine, guanine, and adenine 15- to 30-fold more efficiently than parental cells did. Kinetic analyses revealed that the affinity of the JPA2 transporter for all three purine bases was much greater than that of the wild-type nucleobase transport system. Moreover, nucleobase transport in JPA2 cells, unlike that in parental cells, was insensitive to inhibition by DPA, 4-nitrobenzylthioinosine, sulfhydryl reagents, and nucleosides. No alterations in nucleoside transport capability, phosphoribosylpyrophosphate levels, or purine phosphoribosyltransferase enzymes were detected in JPA2 cells. Thus, JPA2 cells express a novel nucleobase transport capability which can be distinguished from the nucleoside transport function by multiple biochemical parameters.

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

Effect of large targeted deletions on the mitotic stability of an extra chromosome mediating drug resistance in Leishmania.

A mitotically stable linear extra chromosome obtained in a Leishmania donovani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a central inversion region, and is derived from a conservative amplification event of a approximately 140-kb subtelomeric end of chromosome 19. Large-sized targeted deletions of the central region were performed through homologous recombination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb respectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over >140 days. Results differed according to the deletion created. By contrast with the smallest deletion the two largest deletions altered mitotic stability, leading to progressive loss of the size-reduced chromosomes with similar kinetics in both mutants. The 30-kb region common to both deletions may therefore be considered as involved in mitotic stability. A 44-kb contig covering this region could be assembled and sequenced. The analysis of this sequence did not reveal any sequence elements typical of centromeric DNA. By contrast, its enrichment in homopolymer tracts suggests that this region might contain an origin of replication.

Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene.

We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents. In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-beta-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs. The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein. Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein. The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested. The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein. This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine. On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels. We conclude that cellular resistance to the toxicity of 1-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.

Regulation of RNA- and DNA-directed actions of 5-fluoropyrimidines in mouse T-lymphoma (S-49) cells.

The mouse T-lymphoma (S-49) cell line is useful for individually studying RNA- and DNA-directed effects of 5-fluoropyrimidines. On the basis of their metabolic activation, biochemical effects on pyrimidine nucleotide metabolism, and biological toxicity, we hve established that incubation of S-49 cells with 5-fluorodeoxyuridine produces only DNA-directed toxicity (thymidylate synthetase inhibition), incubation with 5-fluorouracil (FUra) + thymidine only RNA-directed toxicity, and incubation with FUra alone produces both DNA- and RNA-directed toxicity. The DNA component of 5-fluoropyrimidine toxicity causes immediate growth inhibition of asynchronous S-49 cell cultures, which is self-limited within 12 hr both by the accumulation of intracellular deoxyuridine 5'-monophosphate competing for thymidylate synthetase binding and by the excretion of deoxyuridine into the cell medium which competes with 5-fluorodeoxyuridine uptake. The RNA-directed component causes growth inhibition and cell kill after a delay of 1 doubling time in asynchronous cultures. Studies with cells synchronized by centrifugal elutriation indicate that the RNA-directed FUra effects are expressed only in the G1 phase of the cell cycle and cause rapid cell lysis, while the DNA-directed component is specific to the S phase. Experiments using continuous exposure of synchronized cells to FUra alone demonstrate that the activities of the RNA- and DNA-directed components interact with each other. Specifically, DNA-directed toxicity arrests cells in S phase, preventing them from progressing into G1 where RNA-directed toxicity is expressed, which may account for the augmentation of FUra toxicity by thymidine as reported in other systems.

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.

Cloning and sequence analysis of a Drosophila melanogaster cDNA encoding IMP dehydrogenase.

A cDNA encoding the entire Drosophila melanogaster IMP dehydrogenase (IMPDH) protein was isolated and sequenced. Translation of the impdh cDNA nucleotide sequence indicated that the Drosophila IMPDH exhibited 48% and 65% amino acid identity to the Leishmania donovani and human isoform II counterparts, respectively. Northern analysis revealed that expression of the 2.4 kb impdh transcript was equivalent in adult Drosophila head and body and that impdh expression was developmentally regulated. In situ hybridization of the cloned impdh cDNA probe to Drosophila salivary gland chromosomes indicated that the impdh gene is located at position 9E1-4 on the X chromosome.

Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells.

The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Plasma neuropeptide Y on admission to a coronary care unit: raised levels in patients with left heart failure.

STUDY OBJECTIVE - The aim of the study was to measure plasma neuropeptide Y, which is related to sympathetic nerve stimulation, in patients admitted to a coronary care unit and to relate the findings to clinical information. DESIGN - Plasma neuropeptide Y was measured on admission and the results were related to the cause of admission and to clinical information collected prospectively and retrospectively. SUBJECTS - Plasma subjects were obtained from 377 consecutive daytime admissions to the coronary care unit at Södersjukhuset. Results of only the first sample in each patient are included in this study, so 45 cases observed more than once (readmitted patients) were omitted. Six samples were abandoned because of technical failures. The study therefore comprises 326 patients. Clinical diagnoses were defined as acute myocardial infarction, arrhythmia, angina pectoris, and miscellaneous (all other diagnoses). Heart failure was defined according to a modified Killip scheme. MEASUREMENTS and RESULTS - Neuropeptide Y like immunoreactivity was measured by radio-immunoassay. Plasma concentrations above normal (greater than 30 pmol.litre-1) were found in association with: increased age, female sex, diuretic treatment, tachycardia, arterial hypotension, increased respiratory rate, and mortality in the unit. There was a strong relationship between high neuropeptide Y concentrations and: moderate left heart failure (63%), pulmonary oedema (90%), and cardiogenic shock (100%). Of patients without heart failure only 25% had raised neuropeptide Y. In multivariate analysis, the severity of heart failure (Killip class), heart rate and respiratory rate were the only variables that were significantly and independently related to plasma neuropeptide Y. CONCLUSIONS - The presence and degree of circulatory disturbance, in particular tachycardia and left heart failure, were strongly related to increased plasma concentrations of neuropeptide Y in coronary care patients.

Nucleoside transporters of parasitic protozoa.

Protozoan parasites are incapable of synthesizing purine nucleotides de novo and so must salvage preformed purines from their hosts. This process of purine acquisition is initiated by the translocation of preformed host purines across parasite or host membranes. Here, we report upon the identification and isolation of DNAs encoding parasite nucleoside transporters and the functional characterization of these proteins in various expression systems. These potential approaches provide a powerful approach for a thorough molecular and biochemical dissection of nucleoside transport in protozoan parasites.

Nutritional requirements of wild-type and folate transport-deficient Leishmania donovani for pterins and folates.

The nutritional requirements for folates and pterins were assessed for two strains of Leishmania donovani promastigotes, a wild-type (D1700) parental strain and a mutant derivative (MTXA5) which possesses a markedly diminished capacity to transport both [3H]folate and [3H]methotrexate (MTX). Both L. donovani strains have an absolute growth requirement for an exogenous pterin, since their proliferation could not be sustained in completely defined medium lacking either a pterin or a folate. Supplementation of the growth medium by many of a spectrum of folates and pterins could support growth of the wild-type cell line. Surprisingly, however, the MTXA5 strain could not thrive in folate-deficient medium fortified with any of the pterins that promoted wild-type cell division, including biopterin and neopterin. The relationship between the incapacity of MTXA5 cells to transport folate and methotrexate and their inability to multiply in folate-deficient medium supplemented with pterins was evaluated using genetic approaches. First, an independently generated MTX-resistant mutant, MTXB4, was isolated in 1 mM MTX. The phenotype of the MTXB4 cells was like that of MTXA5 cells since they were unable to transport [3H]MTX and [3H]folate or grow in folate-deficient medium supplemented with pterins. Second, three revertants of the MTXA5 cells were selected for their ability to grow in 1 microM biopterin. All three revertants had regained [3H]folate and [3H]MTX transport capability concomitant with growth sensitivity to methotrexate toxicity. These observations provide genetic evidence that the competence of L. donovani to transport [3H]folate and [3H]MTX and the capability of the organisms to utilize pterins as a nutritional factor are influenced by a common genetic locus.