Quantitation of oxypurines and allopurinol metabolites in biological fluids by cation-exchange chromatography

Chromatography of urine, cerebrospinal fluid, and deproteinized serum on 30 × 0.9 cm columns of AG 50 permits the convenient separation of hypoxanthine, xanthine, allopurinol, and its metabolites. Quantitation by determination of absorbance at 250 mμ is accurate and can be automated. Column preparation is simple, and the columns can be regenerated and used daily for several months with no loss of resolution.     

Novel mutation in the human HPRT1 gene and the Lesch-Nyhan disease

Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel point mutation that led to HGprt-related neurological dysfunction (HND) in a family in which there was a missense mutation in exon 6 of the coding region of the HPRT1 gene: g.34938G>T, c.403G>T, p.D135Y. Molecular diagnosis is consistent with the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.     

Membrane diffusion- and capillary blood volume measurements are not useful as screening tools for pulmonary arterial hypertension in systemic sclerosis: a case control study

Background

Lung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.

Methods

Healthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.

Results

Lavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.

Conclusion

Iron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease.     

The spectrum of mutations causing HPRT deficiency: an update

Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior. Mutations in the same gene also cause less severe clinical phenotypes with only some portions of the full syndrome. A large database of 271 mutations associated with both full and partial clinical phenotypes was recently compiled. Since the original database was assembled, 31 additional mutations have been identified, bringing the new total to 302. The results demonstrate a very heterogeneous collection of mutations for both LND and its partial syndromes. The differences between LND and the partial phenotypes cannot be explained by differences in the locations of mutations, but the partial phenotypes are more likely to have mutations predicted to allow some residual enzyme function. The reasons for some apparent exceptions to this proposal are addressed.     

The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases

In humans, mutations in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) are associated with a spectrum of disease that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. Previous attempts to correlate different types or locations of mutations with different elements of the disease phenotype have been limited by the relatively small numbers of available cases. The current article describes the molecular genetic basis for 75 new cases of HPRT deficiency, reviews 196 previously reported cases, and summarizes four main conclusions that may be derived from the entire database of 271 mutations. First, the mutations associated with human disease appear dispersed throughout the hprt gene, with some sites appearing to represent relative mutational hot spots. Second, genotype-phenotype correlations provide no indication that specific disease features associate with specific mutation locations. Third, cases with less severe clinical manifestations typically have mutations that are predicted to permit some degree of residual enzyme function. Fourth, the nature of the mutation provides only a rough guide for predicting phenotypic severity. Though mutation analysis does not provide precise information for predicting disease severity, it continues to provide a valuable tool for genetic counseling in terms of confirmation of diagnoses, for identifying potential carriers, and for prenatal diagnosis.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

New Pedal Remains of Megaladapis and Their Functional Significance. Am J Phys Anthropol 100:115–139

Erratum: Wunderlich RE, Simons EL, Jungers WL. 1996. New Pedal Remains of Megaladapis and Their Functional Significance. Am J Phys Anthropol 100:115–139.     

The metabolism of 3-deazaguanine and 3-deazaguanosine by human cells in culture

The metabolism of the purine analogs 3-deazaguanine and 3-deazaguanosine was studied in cultured human cells using radiolabeled tracers, individual enzyme assays, and mutant cell lines. The toxicity of each drug appeared to require conversion to the 5' nucleotide. The base was converted to the nucleotide by hypoxanthine guanine phosphoribosyl transferase. The conversion of the nucleoside to the nucleotide was catalyzed by an unidentified kinase. Purine nucleoside 3-deazaguanosine-5'-monophosphate was converted to its corresponding di- and triphosphate by guanylate kinase. Both the base and the nucleoside were incorporated into DNA but not RNA.     

Maternal and fetal placental growth hormone and IGF axis in type 1 diabetic pregnancy

Aim

Placental growth hormone (PGH) is a major growth hormone in pregnancy and acts with Insulin Like Growth Factor I (IGF-I) and Insulin Like Growth Hormone Binding Protein 3 (IGFBP3). The aim of this study was to investigate PGH, IGF-I and IGFBP3 in non-diabetic (ND) compared to Type 1 Diabetic (T1DM) pregnancies.

Methods

This is a prospective study. Maternal samples were obtained from 25 ND and 25 T1DM mothers at 36 weeks gestation. Cord blood was obtained after delivery. PGH, IGF-I and IGFBP3 were measured using ELISA.

Results

There was no difference in delivery type, gender of infants or birth weight between groups. In T1DM, maternal PGH significantly correlated with ultrasound estimated fetal weight (r = 0.4, p = 0.02), birth weight (r = 0.51, p<0.05) and birth weight centile (r = 0.41, p = 0.03) PGH did not correlate with HbA1c.Maternal IGF-I was lower in T1DM (p = 0.03). Maternal and fetal serum IGFBP3 was higher in T1DM. Maternal third trimester T1DM serum had a significant band at 16 kD on western blot, which was not present in ND.

Conclusion

Maternal T1DM PGH correlated with both antenatal fetal weight and birth weight, suggesting a significant role for PGH in growth in diabetic pregnancy.IGFBP3 is significantly increased in maternal and fetal serum in T1DM pregnancies compared to ND controls, which was explained by increased proteolysis in maternal but not fetal serum. These results suggest that the normal PGH-IGF-I-IGFBP3 axis in pregnancy is abnormal in T1DM pregnancies, which are at higher risk of macrosomia.