Oded Sperling

Accelerated erythrocyte 5-phosphoribosyl-1-pyrophosphate synthesis. A familial abnormality associated with excessive uric acid production and gout

Abstract Increased erythrocyte PRPP content and enhanced in vitro erythrocyte PRPP generation was found in two gouty brothers having excessive uric acid production and normal erythrocyte HGPRT activity. The transmission of the metabolic defect in this family is compatible with the recessive X -linked mode of inheritance.

Human erythrocyte phosphoribosylpyrophosphate synthetase mutationally altered in regulatory properties

Abstract A mutant phosphoribosylpyrophosphate synthetase was found in the erythrocytes of a gouty subject with excessive purine production. The mutant enzyme exhibited normal catalytic properties but decreased sensitivity to inhibition by guanosine-5′-diphosphate, adenosine-5′-diphosphate, adenosine-5′-monophosphate and 2,3-diphosphoglyceric acid at physiological phosphate concentration. Selective alteration by mutation of the regulatory properties shows the enzyme to be allosteric. To our knowledge this is the first demonstration in man of an overproduction disease due to excess activity of a regulatory enzyme as a direct effect of mutation.

Protein kinase C‐ε is involved in the adenosine‐activated signal transduction pathway conferring protection against ischemia‐reperfusion injury in primary rat neuronal cultures

Adenosine activates a signal transduction pathway (STP) in the heart and the brain, conferring protection against ischemia‐reperfusion insult. Activation of protein kinase C (PKC), probably mainly PKC‐ε, has been demonstrated to be part of the heart STP, but its role in the neuronal pathway is less clear. Here, we provide proof for the participation of PKC‐ε in the neuronal adenosine‐activated STP. Primary rat neuronal cultures were exposed to chemical ischemia by iodoacetate, followed by reperfusion. The cultured neurons were protected against this insult by activation of the adenosine mechanism, by N6‐(R)‐phenylisopropyladenosine [R(–)‐PIA], a specific A1 adenosine receptor agonist. Exposure of the cultures to bisindolylmaleimide I, a highly selective PKC inhibitor, abrogated the protection. The exposure of the cultures to R(–)‐PIA was found to result in phosphorylation (activation) of PKC‐ε. Furthermore, insertion into the cells of a specific peptide inhibitor of PKC‐ε translocation (εV1‐2), also abrogated the protection conferred by R(–)‐PIA. These results demonstrate that activation of PKC‐ε is a vital step in the neuronal adenosine‐activated STP.

Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures

The effect of opening and of blocking of ATP-sensitive potassium (K(ATP)) channels on the short-term capacity of neurons to resist ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to metabolic poisoning by iodoacetic acid (150 microM, 150 min), followed by reperfusion (1 h). The metabolic poisoning resulted in a marked decrease in cellular ATP content (from 65.3 +/- 13.4 to 21.6 +/- 11.7 nmole/mg protein), simulating an ischemia, or hypoxia-induced condition of energy crisis. The degree of neuronal damage was assessed by the trypan blue exclusion test. Exposure of the neurons to the channel-opener cromakalim (10 microM; 15 min), prior to the insult, induced resistance, which could be abolished by the specific channel blocker glibenclamide (2 microM). Glibenclamide also abolished the protection acquired by preconditioning of the neurons with iodoacetate (IA; 100 microM), the adenosine A1 agonist N6-(R)-phenylisopropyladenosine (R-PIA; 100 microM), or with the protein kinase C (PKC) activator 1,2 dioctanoyl-rac-glycerol (DOG; 1 microM). The results indicate that in the neurons, opening of the K(ATP) channels confers protection against an ATP-depleting crisis, and suggest that the protective effects induced by adenosine and by activation of PKC, are mediated by the opening of these channels.

Oxidative stress activates transcription factor NF-κB-mediated protective signaling in primary rat neuronal cultures

Activation of transcription factor nuclear factor-κB (NF-κB) can result in enhanced de novo synthesis of both proteins that confer protection and those that cause death. The present study was undertaken to clarify in primary neuronal cultures the consequences of the oxidative stress-induced activation of NF-κB and mediation of death or survival signals. The neuronal cultures were exposed to chemical ischemia (iodoacetic acid), followed by reperfusion (I/R insult). This insult injured the neurons, as manifested in a 7- to 10-fold increase in LDH release, and decreased the cellular content of IκBα by 55–65 %, indicating NF-κB activation. The antioxidants LY231617, melatonin, and sodium salicylate and the antioxidant and inhibitor of NF-κB activation pyrrolidine dithiocarbamate, protected the neurons against the insult and prevented the decrease in cellular IκBα content. In contrast, inhibition of NF-κB translocation by SN50 in both uninsulated and insulted neuronal cultures resulted in a 2.9- and 2.4-fold increase in LDH release, respectively. The results indicate that the insult-induced oxidative stress activates transcription factor NF-κB associated with induction of protection and suggest that constitutive activation of NF-κB under physiological conditions acts to protect the neurons against physiological injury.

Uric acid nephrolithiasis.

Uric acid is the end-product of purine nucleotide metabolism in man. The renal handling of urate is a complicated process, resulting in a fractional clearance of 8.2-10.3%. The anhydrous form is thermodynamically the most stable uric acid crystal. Uric acid is a weak acid that ionizes with a Pka at pH 5.75. At the normal acidic region, uric acid solubility is strongly increased by urinary pH. The prevalence of uric acid stones varies between countries, reflecting climatic, dietary, and ethnical differences, ranging from 2.1% (in Texas) to 37.7% (in Iran). The risk for uric acid stone formation correlates with the degree of uric acid supersaturation in the urine, depending on uric acid concentration and urinary pH. Hyperuricosuria is the major risk factor, the most common cause being increased purine intake in the diet. Acquired and hereditary diseases accompanied by hyperuricosuria and stone disease include: gout, in strong correlation with the amount of uric acid excreted, myelo- and lymphoproliferative disorders, multiple myeloma, secondary polycythemia, pernicious anemia and hemolytic disorders, hemoglobinopathies and thalassemia, the complete or partial deficiency of HGPRT, superactivity of PRPP synthetase, and hereditary renal hypouricemia. A common denominator in patients with idiopathic and gouty stone formers is a low urinary pH. Uric acid nephrolithiasis is indicated in the presence of a radiolucent stone, a persistent undue urine acidity and uric acid crystals in fresh urine samples. A radiolucent stone in combination with normal or acidic pH should raise the possibility of urate stones.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum xanthine oxidase in jaundice.

Serum xanthine oxidase activity was measured by a radiochemical method in 137 consecutive patients with jaundice of varying etiology and in 40 non-jaundiced patients with liver or other disease. Serum xanthine oxidase was markedly increased, up to 50 times the upper normal limit (mean + 2 S.D.), in 32 out of 34 patients with infectious hepatitis. A slight elevation of serum xanthine oxidase, up to twice the upper normal limit, was found in 2 out of 49 patients with extrahepatic obstructive jaundice and in 4 out of 20 patients with chronic renal failure. In comparison to serum glutamic-oxaloacetic transaminase and lactate dehydrogenase serum xanthine oxidase appeared to be the more sensitive and specific indicator of acute hepatocellular damage.

Hypouricemia, Hypercalciuria, and Decreased Bone Density: A Hereditary Syndrome

Genetically determined hypouricemia in man due to increased renal urate clearance is usually associated with additional renal tubular defects (1–3). Renal hypouricemia due to an isolated renal tubular defect has also been described but appears to be a rare condition (4,5). We report a new genetically determined syndrome in man, in which the renal hypouricemia is associated with idiopathic hypercalciuria and decreased bone density.

The effect of ribose 5-phosphate and 5-phosphoribosyl-1-pyrophosphate availability on de novo synthesis of purine nucleotides in rat liver slices.

The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice.

Abstract : Lesch‐Nyhan syndrome is a pediatric metabolic‐neurological syndrome caused by the X‐linked deficiency of the purine salvage enzyme hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT‐deficient transgenic mice. The HGPRT‐deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT‐deficient cells. The following abnormalities were found : absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides ; 27.8% increase in the availability of 5‐phosphoribosyl‐1‐pyrophosphate ; 9.4‐fold acceleration of the rate of de novo nucleotide synthesis ; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine) ; enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2‐fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT‐deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch‐Nyhan syndrome.