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Biomedical subjects

N Blau

Publications and source records attributed to N Blau.

At least 91 records · Page 5Linked to original sources
PubMed1995

Characterization of the human PCBD gene encoding the bifunctional protein pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor for the transcription factor HNF-1 alpha.

The human pterin-4 alpha-carbinolamine dehydratase (PCD)/dimerization cofactor for the transcription factor HNF-1 alpha is a bifunctional protein proposed to be involved in entirely different biochemical functions. We previously established the complete amino acid sequence for the human liver PCD and subsequently isolated its corresponding cDNA. Using this cDNA as a probe, we isolated and determined the complete nucleotide sequence and flanking regions of the single human PCBD gene. The protein coding region of the gene is about 5 kb in length and contains 4 exons. We also defined the messenger RNA 5'-end by reverse transcription of the cap structure, thus allowing to analyze the promoter organization. Within the 5'-flanking sequence, potential regulatory regions include consensus binding sites for transcription factor Sp1, an AP-1, and several AP-2 binding sites; however, the 5' upstream region lacks both a proximal TATA and CAAT box promoter element.

Amino Acid Sequence↗
PubMed1995

Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning approximately 30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5'-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

Amino Acid Sequence↗
PubMed1995

Comparison of C-reactive protein and white blood cell count with differential in neonates at risk for septicaemia.

UNLABELLED: We prospectively compared the diagnostic value of C-reactive protein (CRP) and white blood cell counts for detection of neonatal septicaemia. Sensitivity and specifity in receiver operating characteristics, and positive and negative predictive value of CRP and white blood cell count were compared in 195 critically ill preterm and term newborns clinically suspected of infection. Blood cultures were positive in 33 cases. During the first 3 days after birth CRP elevation (sensitivity 75%, specifity 86%), leukopenia (67%/90%), neutropenia (78%/80%) and immature to total neutrophil count (I/T) ratio (78%/73%) were good diagnostic parameters, as opposed to band forms with absolute count (84%/66%) or percentage (79%/71%), thrombocytopenia (65%/57%) and toxic granulations (44%/94%). Beyond 3 days of age elevated CRP (88%/87%) was the best parameter. Increased total (84%/66%) or percentage band count (79%/71%) were also useful. Leukocytosis (74%/56%), increased neutrophils (67%/65%), I/T ratio (79%/47%), thrombocytopenia (65%/57%) and toxic granulations had a low specifity. The positive predictive value of CRP was 32% before and 37% after 3 days of age, that of leukopenia was 37% in the first 3 days. CONCLUSION: During the first 3 days of life CRP, leukopenia and neutropenia were comparably good tests while after 3 days of life CRP was the best single test in early detection of neonatal septicaemia. Serial CRP estimations confirm the diagnosis, monitor the course of infection and the efficacy of antibiotic treatment.

C-Reactive Protein↗
PubMed1995

A missense mutation in a patient with guanosine triphosphate cyclohydrolase I deficiency missed in the newborn screening program.

A patient with guanosine triphosphate cyclohydrolase I deficiency passed the newborn phenylketonuria screening program. The characteristic clinical phenotype developed in a 5-month-old patient; elevated plasma phenylalanine, undetectable urinary pterins, and absence of the enzyme activity in a liver biopsy were present. A point mutation that results in an amino acid substitution from methionine to isoleucine at position 211 was proposed to be the cause for this new phenotypic expression of guanosine triphosphate cyclohydrolase I deficiency.

Base Sequence↗
PubMed1995

Nitric oxide and infection: another view.

Nitric oxide (NO) has been nicknamed "murderer" and "mediator" because it has toxic and signaling properties. We review these two aspects of NO synthesis from the perspective of the clinical infectious disease specialist by considering the potential of NO as an endothelium-derived relaxing factor (EDRF) in inflammation and sepsis and its potential as an antimicrobial system. We deviate from observations in recent authoritative reviews and point to important species differences that make it unlikely that NO serves as an EDRF mediating inflammatory vasodilatation in humans or that NO synthesized by humans phagocytes has an antimicrobial function. We propose that in humans, No synthesis is more confined and compartmentalized than in certain other animal species, and therefore, unwelcome toxicity, vasodilatation, or disturbance of paracrine signaling mechanisms (i.e., modulation of phagocytic cell functions) are avoidable during inflammation.

Animals↗
PubMed1995

Efficacy of oral citrate administration in primary hyperoxaluria.

Urinary citrate is a potent inhibitor of calcium oxalate (CaOx) crystallization, but oral citrate has rarely been used in patients with primary hyperoxaluria (PH). We studied the effect of sodium citrate administration (0.1-0.15 g/kg/day) on urinary citrate excretion and CaOx saturation in seven paediatric patients and the clinical response to long-term treatment (average 4 years) in five patients. Urinary citrate increased from 0.73 to 2.54 mmol/24 h/1.73 m2 and urinary saturation for CaOx (calculated by equil 2) decreased from 11.41 to 6.79 (for both, p < 0.02). Long-term administration of alkali citrate [0.15 g (0.5 mmol)/kg/day] resulted in stable or improved renal function in three and slow deterioration in two partially non-compliant patients. Alkali citrate is effective in patients with PH.

Administration, Oral↗
PubMed1994

Tetrahydrobiopterin as another EDRF in man.

Endotoxin and inflammatory cytokines downregulate expression of constitutive nitric oxide synthase (cNOS) in human vascular endothelial cells with concomitant increase of tetrahydrobiopterin synthesis in these cells and parallel upregulation of inducible NOS expression in smooth muscle cells, indicating compartmentalized nitric oxide (NO) production under septic conditions in man. In this report the compartmentalization has been further studied using dual chamber cell cultures with inflammatory activated human endothelial cells. We show that endothelial cells secrete BH4 vectorially into the basal direction thereby providing underlining smooth muscle cells with the cofactor necessary for NO production. Furthermore, by laser Doppler velocimetry we show that intraarterial infusion of BH4 induces strong vasodilatation in man. Consumption of L-arginine and production of cyclic GMP increased and therefore imply NO as second messenger. Thus the discovery of an endothelium-derived factor regulating NOS activity would reconcile the concept of an inflammatory EDRF that is not NO itself but results in NO-dependent vasodilatation in man.

Animals↗
PubMed1994

Tetrahydrobiopterin is a secretory product of murine vascular endothelial cells.

Tetrahydrobiopterin (BH4) is an obligatory cofactor of nitric oxide synthase and an essential regulator of its activity. The murine vascular endothelial cell line send1 constitutively secretes large amounts of BH4 as well as scant amounts of neopterin, an oxidized intermediate in the de novo biosynthesis of BH4. Further enhancement of BH4 and neopterin secretion is achieved by activation with endotoxin (LPS) and interferon-gamma. This finding is in accordance with previous described BH4 secretion by human endothelial cells. It supports the view that endothelial cells are the source of BH4 serving vascular needs in vivo. In septic conditions, BH4 released by endothelial cells in large amounts could serve induced nitric oxide synthase in smooth muscle cells, thereby acting as another endothelium-derived relaxing factor mediating vasodilatation.

Animals↗
PubMed1994

Nitric oxide production depends on preceding tetrahydrobiopterin synthesis by endothelial cells: selective suppression of induced nitric oxide production by sepiapterin reductase inhibitors.

Using murine vascular endothelial cells expressing both constitutive and inducible nitric oxide synthases (cNOS and iNOS), we explored the feasibility of suppressing cytokine-induced nitric oxide (NO) production without affecting constitutive NO production by inhibition of the tetrahydrobiopterin (BH4) biosynthesis. We show in this study that in endothelial cells cytokine/endotoxin-activated BH4 synthesis precedes the induction of NO generation. Using the sepiapterin reductase inhibitors phenprocoumon or dicumarol as BH4 synthesis inhibitors, we achieved a pronounced and selective suppression of induced NO production in cytokine-activated endothelial cells. Addition of exogenous BH4, but not sepiapterin, restored NO production in the presence of the inhibitors. Despite profound inhibition of the BH4 biosynthesis, constitutive NO synthesis was not affected, thereby demonstrating the selectivity and specificity of the inhibitors. Suppression of enhanced NO production by sepiapterin reductase inhibitors such as cumaroles could provide pharmacologic means for therapeutic interventions in NO-mediated pathophysiologic events.

Alcohol Oxidoreductases↗
PubMed1994

Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity.

6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent Km for the substrate dihydroneopterin triphosphate (8.5 microM for the human and 8.0 microM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.

Alcohol Oxidoreductases↗
PubMed1994

Possible high frequency of tetrahydrobiopterin deficiency in south Brazil.

We report our experience with the deficiency of 6-pyruvoyltetrahydropterin synthase, the most common form of tetrahydrobiopterin deficiency. We investigated 5200 patients suspected of having some inborn error of metabolism in a 10-year period, and detected 30 cases (from 28 sibships) of hyperphenylalaninaemias, HPA. From these, 4 sibships (5 patients) were affected by deficiency of 6-pyruvoyltetrahydropterin synthase. All of them were ethnically mixed, with some European ancestry detected in all. The age of diagnosis ranged from 2 to 9 years, and all were initially referred for investigation by having mental retardation and seizures. All of them showed low urinary biopterin levels and a marked elevation of neopterin. Although we detected only a few cases of HPA (30), 5 cases of 6-pyruvoyltetrahydropterin account for almost 20% of this total. The literature, however, reports a proportion of around 0.5%. As the frequency of classical phenylketonuria in our region is similar to that found in Caucasians (1/12,500), we believe that the frequency of this disease in South Brazil may be higher than expected (of the order of 1/400,000). We speculate that this finding could be related to a genetic drift (or founder effect).

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

Antenatal diagnosis of tetrahydrobiopterin deficiency by quantification of pterins in amniotic fluid and enzyme activity in fetal and extrafetal tissue.

Prenatal diagnosis of tetrahydrobiopterin (BH4) deficiency was undertaken by evaluating the pterin patterns in amniotic fluid and the specific enzyme activities in fetal or extrafetal tissues. This allowed the prenatal diagnosis in 19 pregnancies at risk. In 8 families with a child already affected by dihydropteridine reductase deficiency 4 fetuses were diagnosed as homozygotes and 4 as heterozygotes for the defect. In 11 families with a child affected by 6-pyruvoyl tetrahydropterin synthase deficiency 4 fetuses were homozygous, 4 heterozygous and 3 normal. This study also advanced our knowledge of tetrahydrobiopterin metabolism during fetal development. The key enzymes involved in the biosynthesis of BH4 are expressed early and allow the fetus to be autotrophous for its cofactor requirement. In a twin pregnancy, both fetuses were diagnosed to be heterozygotes for dihydropteridine reductase deficiency and primapterin (7-biopterin) in amniotic fluid was increased. This indicates that pterin-4 alpha-carbinolamine dehydratase activity seems to be differently expressed during fetal life. As a consequence, pterins detected in amniotic fluid are of fetal origin and 6- and 7-substituted pterins can be present in amniotic fluid in higher proportions when compared with other body fluids.

Alcohol Oxidoreductases↗
PubMed1994

Effect of high-protein meal plus aspartame ingestion on plasma phenylalanine concentrations in obligate heterozygotes for phenylketonuria.

The effect of a protein-rich meal alone or in combination with 85 mumol/kg body weight aspartame (APM) on plasma phenylalanine and large neutral amino acids (LNAA) was evaluated in obligate heterozygotes for phenylketonuria (PKU) and normal subjects (controls). Thirteen PKU heterozygotes (seven women, six men) and 13 controls (five women, eight men) ingested a 12-noon meal providing approximately 303 mumol/kg Phe. In addition, 10 PKU heterozygotes (five women, five men) and 10 controls (five women, five men) ingested the same meal with 85 mumol/kg APM (providing 75 mumol/kg Phe). Plasma amino acids were analyzed at baseline (-4 and 0 hours) and at 1, 3, and 20 hours after the meal or meal plus APM. Compared with the meal alone, ingestion of the meal plus APM significantly increased plasma Phe concentrations in both controls and PKU heterozygotes. Mean plasma Phe values were higher for controls at 1 hour (95 +/- 7 mumol/L) and for PKU heterozygotes at 3 hours (153 +/- 21 mumol/L). After the addition of APM to the meal, the highest mean plasma Phe concentration was only slightly greater than the usual postprandial range for both controls and PKU heterozygotes. Ingestion of the meal did not increase the plasma Phe/LNAA ratio in either controls or PKU heterozygotes. Compared with baseline, the plasma Phe/LNAA ratio increased significantly 1 hour after combined ingestion of the meal plus APM in both groups (P = .020 and P = .008, respectively); however, the ratios were well below the range of Phe/LNAA values in individuals with mild hyperphenylalaninemia, who are clinically normal and do not require a Phe-restricted diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase.

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.

Alcohol Oxidoreductases↗
PubMed1993

Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelium-derived relaxing factor.

Endothelial cells, through soluble mediators, play an important role in the regulation of the vascular tone. In the present paper we investigated whether endothelial tetrahydrobiopterin (BH4), an obligatory cofactor of nitric oxide (NO) synthase, could serve as such a regulatory mediator. By studying the human vascular endothelial hybrid cells EA hy 926, we found that 1) BH4 biosynthesis is highly regulated (70-fold) by activating and deactivating cytokines; that 2) up to 90% of the induced BH4 is released by activated endothelium, and 3) while intracellular BH4 could be related to cyclic GMP concentrations within the endothelial cells, the bulk of BH4 (up to 90 pmol/10(6) cells) appears not to serve endothelial cell requirements. Activation and deactivation of BH4 synthesis by cytokines was paralleled by other endothelial cell responses reflecting their activity. We propose that BH4 serves as an endothelial mediator augmenting the activity of cytokine-inducible NO synthase in vascular smooth muscle cells. BH4 could thereby account for endothelium-derived relaxing factor activity.

Biopterins↗