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1.  4-Pyridone-3-carboxamide-1-β-D-ribonucleoside Triphosphate (4PyTP), a Novel NAD+ Metabolite Accumulating in Erythrocytes of Uremic Children: A Biomarker for a Toxic NAD+ Analogue in Other Tissues? 
Toxins  2011;3(6):520-537.
We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD+ metabolites (nicotinamide, N1-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N1-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD+ from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD+ analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD+ analogue that inhibits IMP dehydrogenase in other cells.
PMCID: PMC3202843  PMID: 22069723
uremia; erythrocytes; pyridone; NAD+; 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP); nicotinamide riboside (NR); IMP dehydrogenase; HPLC
3.  Synthesis of Genomic and Subgenomic RNA in Mosquito Cells Infected with Two Sindbis Virus nsP4 Mutants: Influence of Intracellular Nucleoside Triphosphate Concentrations▿  
Journal of Virology  2008;82(14):6880-6888.
Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.
PMCID: PMC2446979  PMID: 18508899
4.  A Mutant of Sindbis Virus Which Is Able To Replicate in Cells with Reduced CTP Makes a Replicase/Transcriptase with a Decreased Km for CTP 
Journal of Virology  2004;78(18):9645-9651.
We reported earlier the isolation and characterization of a Sindbis virus mutant, SVPZF, that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SVCPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SVPZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SVCPC was selected for its ability to grow in cells in which only the level of CTP was reduced. Although SVPZF was cross-resistant to CPC, SVCPC was not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SVCPC-infected mosquito cells had a lower Km for CTP (but not for UTP) than did the enzyme made in SVSTD-infected mosquito cells. SVPZF and SVCPC represent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.
PMCID: PMC515026  PMID: 15331697
6.  Use and abuse of allopurinol. 
PMCID: PMC1246665  PMID: 3607420
7.  Central nervous system dysfunction and erythrocyte guanosine triphosphate depletion in purine nucleoside phosphorylase deficiency. 
Archives of Disease in Childhood  1987;62(4):385-391.
Developmental retardation was a prominent clinical feature in six infants from three kindreds deficient in the enzyme purine nucleoside phosphorylase (PNP) and was present before development of T cell immunodeficiency. Guanosine triphosphate (GTP) depletion was noted in the erythrocytes of all surviving homozygotes and was of equivalent magnitude to that found in the Lesch-Nyhan syndrome (complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency). The similarity between the neurological complications in both disorders indicates that the two major clinical consequences of complete PNP deficiency have differing aetiologies: neurological effects resulting from deficiency of the PNP enzyme products, which are the substrates for HGPRT, leading to functional deficiency of this enzyme. immunodeficiency caused by accumulation of the PNP enzyme substrates, one of which, deoxyguanosine, is toxic to T cells. These studies show the need to consider PNP deficiency (suggested by the finding of hypouricaemia) in patients with neurological dysfunction, as well as in T cell immunodeficiency. They suggest an important role for GTP in normal central nervous system function.
PMCID: PMC1778361  PMID: 2439024
8.  A controlled study of diet in patients with gout. 
Annals of the Rheumatic Diseases  1983;42(2):123-127.
To determine whether patients with gout have a diet which is distinctive in quality or quantity a careful dietary questionnaire was posed over 7 days to 61 men with gout and 52 control subjects. The average daily intake of most nutrients, including total purine nitrogen, was similar except that the patients with gout drank significantly more alcohol. Beer was the most popular beverage, and 25 (41%) of those with gout consumed more than 60 g alcohol daily (equivalent to 2 . 5 litres of beer). The heavy drinkers had a significantly higher intake of purine nitrogen, half of which was derived from beer. Though the effect of ingested purine on the blood uric acid is difficult to estimate, it probably was sufficient to have a clinical effect, augmenting the hyperuricaemic effect of alcohol itself.
PMCID: PMC1001083  PMID: 6847259
11.  Allopurinol treatment and its effect on renal function in gout: a controlled study. 
Fifty-nine patients with primary gout were treated with either a combination of colchicine and allopurinol or colchicine alone. Assessments of renal function over 2 years revealed a statistically significant fall of glomerular filtration rate an urine concentrating ability in those receiving only colchicine. The renal function of patients given allopurinol did not change. Treatment with allopurinol resulted ina significant reduction of ammonium excretion, a phenomenon which could not be readily explained. Urate clearance also declined during allopurinol treatment, and the impaired urate clearance associated with gout became more evident. The most important observation was that allopurinol retarded an apparent decline of renal function. Presumably this was achieved through its hypouricaemic effect and implies that the hyperuricaemia of gouty patients is deleterious to the kidneys.
PMCID: PMC1000865  PMID: 7039523
12.  Renal impairment and gout. 
Annals of the Rheumatic Diseases  1980;39(5):417-423.
A study of renal function of 51 patients with gout and an equal number of normouricaemic controls revealed significant differences. A relative impairment of the glomerular filtration rate and urine concentrating ability in the gouty subjects could not be wholly explained on the basis of aging or hypertension. Renal dysfunction was generally mild and was not associated with specific clinical characteristics higher levels of uric acid excretion, or hypertriglyceridaemia. Gout patients excreted urine with a significantly lower pH. This was associated with a relatively high excretion of titratable acid and a deficit of ammonium excretion, which was accentuated by ingestion of an acid load. Urate clearance was significantly reduced in gout, even when expressed as a fraction of the glomerular filtration rate.
PMCID: PMC1000578  PMID: 7436572
13.  Hypoxanthine guanine phosphoribosyl transferase deficiency presenting with gout and renal failure in infancy. 
Archives of Disease in Childhood  1983;58(10):831-833.
A month old infant had gout and renal failure caused by hypoxanthine guanine phosphoribosyl transferase (HGPRTase) deficiency. Investigations showed a high uric acid value, crystal nephropathy on ultrasound, and uric acid deposition on renal biopsy. The HGPRTase value was low in red cells and fibroblasts.
PMCID: PMC1628265  PMID: 6639135
14.  Tienilic acid: a single treatment for hyperuricaemia and hypertension? 
Annals of the Rheumatic Diseases  1980;39(4):373-376.
Tienilic acid is a drug with established uricosuric and hypotensive properties. We have examined its potential role as a single treatment for hyperuricaemia and hypertension, 2 disorders which are commonly associated. In 17 subjects with gout, blood uric acid levels were reduced by approximately 50%. Eleven of these patients also had hypertension which was improved by tienilic acid. However, a statistically significant effect was observed only with standing diastolic blood pressure. Side effects included acute episodes of gout in 4 patients and transient loin pain and dysuria in 1 patient. The precipitation of gouty arthritis is an acknowledged risk of all potent hypouricaemic drugs. The hazard of urate deposition in the renal tract implicit in the history of loin pain is a more serious complication. Thus, it would appear that tienilic acid is a potentially valuable drug which may have a special role in patients with hyperuricaemia and hypertension. Further study is necessary to determine how it may be best administered without the risk of renal damage.
PMCID: PMC1000559  PMID: 7436564
15.  Mechanism and treatment of hypertriglyceridaemia in gout. 
Using the Intralipid lipid tolerance test we could not demonstrate any direct effect of serum triglyceride on uric acid or any influence of hyperuricaemia on triglyceride removal. This result supports previous studies suggesting that hyperuricaemia and hypertriglyceridaemia are linked through the association of obesity and alcohol excess rather than a direct cause and effect mechanism. It was possible to demonstrate significant reductions of serum triglyceride in patients with gout by reducing either their alcohol intake or body weight. Reduction of serum uric acid by probenecid had no effect on serum triglyceride or cholesterol. Similarly, allopurinol had no significant effect on serum triglyceride, but a significant fall of serum cholesterol was observed.
PMCID: PMC1000314  PMID: 434944
16.  Uric acid, gout and the kidney. 
Journal of Clinical Pathology  1981;34(11):1245-1254.
PMCID: PMC494475  PMID: 7320221
17.  Familial gout and renal failure. 
Archives of Disease in Childhood  1981;56(9):699-704.
Clinical gout and renal failure was seen in a 9-year-old girl. The family tree showed that 9 out of 11 young females in three generations suffered from hyperuricaemia and normal (n = 1), or impaired (n = 8), renal function. One set of twins occurred in each generation and there is only one living male subject. In members with renal failure there was no improvement in renal function after treatment of hyperuricaemia, and in 2 sisters oral contraceptives appeared to precipitate hypertension. This clinical picture may be more common than is generally realised because of failure to compare blood uric acid values with suitable age- and sex-matched controls. The evidence from this family suggests that hyperuricaemia preceded the development of renal failure.
PMCID: PMC1627300  PMID: 7294873
18.  Complete deficiency of adenine phosphoribosyltransferase: a third case presenting as renal stones in a young child. 
We report a third case of 2, 8-dihydroxyadenine stones in a child with a complete lack of the adenine salvage enzyme--adenine phosphoribosyltransferase (APRT). The propositus, a 20-month-old girl of consanguineous Arab parents, presented with multiple urinary tract infections and supposed 'uric acid' stones in the right renal pelvis and left ureter. Both parents and one brother were heterzygotes for the defect, in keeping with an autosomal recessive mode of inheritance. In contrast with the other purine salvage enzyme disorder of childhood with true uric acid stones (the Lesch-Nyhan syndrome), uric acid excretion was normal in all family members. As in our previous case, treatment with allopurinol, without alkali, has eliminated the urinary excretion of 2, 8-dihydroxyadenine: the stones were removed surgically. 2, 8-Dihydroxyadenine should be considered in any child thought to have uric acid stones and tests made to distinguish the two compounds.
PMCID: PMC1545198  PMID: 420519

Results 1-24 (24)